induction of immunological tolerance in the pig-to … ian patrick joseph.pdf · the inclusion...

INDUCTION OF IMMUNOLOGICAL TOLERANCE IN THE

PIG-TO-BABOON XENOTRANSPLANTATION MODEL

Studies aimed at achieving mixed hematopoietic chimerism and preventing

associated thrombotic complications

ISBN 90-9014712-8

© I.PJ. Alwayn. All rights reserved. No part of this dissertation may be reproduced, stored in a retrieval of any nature, or transmitted in any form by any means, electronic, mechanical, photocopying, recording or otherwise, without the permission of the author.

Printed by Pasmans Offsetdrukkerij'b.v., Den Haag

INDUCTION OF IMMUNOLOGICAL TOLERANCE IN THE

PIG-TO-BABOON XENOTRANSPLANTATION MODEL

Studies aimed at achieving mixed hematopoietic chimerism and preventing

associated thrombotic complications

DE INDUCTIE VAN IMMUNOLOGISCHE TOLERANTIE IN HET

V ARKEN-NAAR-BA VlAAN XENOTRANSPLANTA TIE MODEL

Studies gericht op het bereiken van gemengd hematopoietisch chimerisme en de

preventie van geassocieerde thrombotische complicaties

PROEFSCHRIFT

Tef verkrijging van de graad van doctor

aan de Erasmus Universiteit Rotterdam

op gezag van de Rector Magnificus

Prof.dr.ir. J.H. van Bemmel

en volgens besluit van het College vocr Promoties

de open bare verdediging zal piaatsvinden op

woensdag II april 2001 om 15.30 uur

door

Ian Patrick Joseph Alwayn

geboren te Leidschendam

PROMOTIECOMMISSIE

Promotoren:

Overige leden:

Copromotor:

Prof.dr. l. leeke!

Prof.dr. D.K.C. Cooper

Prof.dr. R. Benner

Prof.dr. F.G. Grosve!d

Prof.dr. W. Weimar

Dr. J.N.M. IJzermans

The studies presented in this dissertation were performed at the Transplantation Biology

Research Center of Massachusetts General Hospital/Harvard Medical School, Boston,

U.S.A.

The work was financially supported by grants from the 'Ter Meulen Fund' of the Royal

Dutch Academy of Arts and Sciences and from the Professor Michael van Vloten Fund.

Voor mijn moeder,

In memory of my father

Contents

CONTENTS

Section I

Chapter 1

Section II

Chapter 2

Introduction

Introduction ~ aims of this dissertation

Attempts to induce mixed hematopoietic chimerism in

pig-to-baboon xenotransplantation

Depletion of macrophages in baboons delays the clearance

of mobilized pig leukocytes

Section III The importance of suppressing xenoreactive antibody

production

Chapter 3

Chapter 4

Section IV

Chapter 5

Chapter 6

Chapter 7

Current and novel methods of depletion and/or suppression

of production of anti-pig antibodies

Effects of specific anti-B and/or anti-plasma cell immuno

therapy on xenoreactive antibody production in baboons

Understanding and preventing the thrombotic complications

associated with pig-to-baboon xenotransplantation

Coagulation and thrombotic disorders associated with

xenotransplantation

Mechanisms of thrombocytopenia following xenogeneic

hematopoietic progenitor cell transplantation

Immunosuppressive therapies and platelet aggregation in

baboons

11

35

59

83

115

137

155

Contents

Chapter 8

Section V

Chapter 9

Chapter 10

Pharmacologic inhibition of platelet aggregation in baboons

Discussion

General discussion

Summary in Dutch

List of abbreviations

List of publications

Acknowledgements

Curriculum vitae auctoris

179

203

213

223

225

231

233

I Introduction

1 Introduction - aims of this dissertation

Adapted from: Alwayn IPJ, Buhler L, Basker M, and Cooper DKC. Immunomodulation strategies in xenotransplantation. In: Current and Future Immunosuppressive therapies following Transplantation. Sayegh M and Remuzzi G (Eds.). In press.

Section 1- Chapter 1

l.l THE NEED FOR XENOTRANSPLANTATION

The outcome of clinical organ transplantation has dramatically improved since the

introduction of cyclosporine (CyA) in 1979 and of other, more recently introduced,

immunosuppressive agents such as azathioprine, mycophenolate mofetil, tacrolimus

and sirolimus. Furthermore, due to more refined surgical techniques and peri operative

management, prolonged survival of allografts is achieved. Due to its relative success,

the inclusion criteria for potential organ transplant recipients have been broadened,

resulting in an even greater shortage of donor organs. The number of patients with

end-stage organ failure that die awaiting organ transplantation is steadily increasing,

mainly because of the shortage of appropriate donor organs (1,2,3). Many clinicians

and investigators believe that strategies directed at expanding the allogeneic donor

pool will not be sufficient to resolve this problem of organ shortage~ In contrast,

successful xenotransplantation - the transplantation of tissues and/or organs between

two different species - has the potential of providing an unlimited supply of donor

organs that would be available when required (4). However, major immunological

barriers, as well as other important issues, at present prevent the implementation of

xenotransplantation clinically (5).

Concordant xenografts - tissues/organs transplanted between phylogenetically close

species, i.e., mouse and rat, monkey and baboon, or nonhuman primate and human -

reject more vigorously than allografts. The mechanism of rejection is thought to be

mediated by a combination of humoral and cellular factors. Conventional

immunosuppressive therapy carmot guarantee long-term graft survival because of the

potent induced antibody response in concordant xenotransplantation. However, with

judicious pharmacological immunosuppressive therapy, the reported sunrival of

concordant heart grafts in nonhuman primates exceeds 500 days (6).

Discordant xenotransplantation - xenotransplantation between phylogenetically

widely disparate species, i.e., guinea pig and rat, pig and baboon, or pig and human -

is characterized by the occurrence of hyperacute rejection (HAR) (7, 8). This type of

12

Introduction

rejection, mediated in primates by preformed (natural) antibodies (directed against the

Galal-3Gal (a-Gal) epitopes) (9), which activate complement, takes place within

minutes to hours after transplantation (10). Histological examination of these rejected

grafts reveals intravascular thrombosis and interstitial hemorrhage (11,12).

It has recently become possible to overcome HAR through antibody and/or

complement depletion or by utilizing organs from pigs transgenic for human

complement regulatory proteins (8, 13, 14, 15). If HAR is successfully prevented, a

delayed type of rejection termed acute humoral xenograft rejection (AHXR) occurs

within days. Cellular infiltrates are invariably found throughout the xenograft,

consisting of macrophages, NK cells, and monocytes (16, 17). Data suggest that this

type of rejection is most probably antibody-mediated but complement-independent. It

has thus far not been possible to overcome AHXR in the pig-to-primate modeL

Survival of xenografts to date has generally been less than 30 days (I8, 19), and has

not exceeded 99 days (\\t'hite DJG, personal communication).

These major immunological differences notwithstanding, it is widely thought that the

pig will be the most appropriate organ source (20, 21), as pigs have a number of

advantages over nonhuman primates as a potential source of organs for humans

(Table 1). Furthermore, pigs can be bred in specific pathogen-free environments, thus

eliminating to a large extent the risk of transfer of disease from pig to human. Certain

herds of miniature swine have fully documented :MHC antigens, which may facilitate

certain manipulations aimed at the induction of tolerance in the recipient, and pigs

may be genetically modified to become less immunogenic to humans. Finally, since

pigs are currently being bred for human consumption, it is likely that the general

public will be amenable to using pigs for human organ xenotransplantation.

Many studies have been directed at attempting to understand the mechanisms of pig

to-nonhuman primate xenograft rejection. Much progress in this field has been made

in the last decade, although the pathophysiology of xenograft rejection has not yet

been fully elucidated. This chapter will briefly summarize our current knowledge of

the mechanisms involved in discordant xenograft rejection and describe the

13


techniques being explored to prevent rejection from occurring. Emphasis will be

placed on our experience in the pig-to-nonhuman primate preclinical model. Various

RELATIVE ADVANTAGES A~D DlSADVA~T AGES OF PIGS AI\D BABOO]'l;S AS

POTE~TIAL SOURCES OF ORGANS A]'I;O TISSUES.

Baboon !!g

Availability Restricted Unlimited

Breeding potential Poor Good

Reproductive age 3-5 years 4-8 months

Length of pregnancy 173-193 days 114 +/. 2 days

Number of orupring 1-2 5-\2

Rate of growth Slow Rapid

Size of adult Qrgaus Inadequate Adequate

Maintenance cost High Low

Similarity

Anatomical Similar Similar

Physiological Very similar Less similar

Immunological Close Distant

Knowledge of tissue typing Limited Considerable

(in selected herds)

Expcrience with genetic engineering None Considerable

Risk of transfer of infection (xenozoonosis) High Low

Availability of specific pathogen-free animals No y~

Public opinion l'v1ixed More favorable

methods of immunomodulation will be discussed, including i) those that deplete or

inhibit complement, ii) phannacological immunosuppressive therapy aimed at

suppression of both T and B cell function, iii) genetic engineering of the donor

animal, and iv) the induction oftolerance and/or accommodation.

14

Introduction

1.2 REJECTION OF XENOGRAFTS

Hyperacute Rejection

HAR is an antibody-dependant, complement-mediated process that occurs within

minutes to hours after revascularization of the transplanted discordant xenograft.

Perper and ~"JJJlan were among the first to describe this process in 1966 in the pig

to-dog model (7). All nonprimate mammals, including the pig, express terminal

Galal·3Gal oligosaccharides (aGal) as surface antigens on many ceUs. including the

vascular endothelium (22, 23). In 1984 Galili et al. reported that humans possess

natural antibodies with anti-aGal specificity (24), but it was not until 7 years later that

Good et al. demonstrated that these antibodies (anti·aGal) playa major role in the

HAR of pig xenografts in primates (9). Anti-aGal are of IgG. IgM. and IgA

~'·;h:1as':-,.::::- ... l,,! '.'"'-·~)um for m .... 'H: .. :Uil ~j->'; o-F ~irculating natural anti-pig antibody in

humans and 100% in baboons (24, 25. 26, 27, 28). IgM is thought to be the major

contributor to HAR (29). Binding of these antibodies to the aGal epitopes on porcine

tissue results in activation of complement through the classical pathway. This initiates

a chain-reaction involving multiple complement components and leads to the

formation of complexes, known as the membrane attack complex of complement,

which causes lysis and opsonisation of cell membranes, resulting in rapid graft

destruction.

Acute Vascular Rejection

Several methods have been developed to successfully prevent HAR, including

extracorporeal immunoadsorption (EIA) of anti-aGal antibody, complement depletion

or inhibition, and/or the use of pigs transgenic for human complement regulatory

proteins. These approaches are discussed below. Once HAR has been averted,

however, acute vascular rejection (AHXR) develops, most probably as a result of the

development of high levels of high-affinity antibody directed against aGal and other

pig epitopes (30). IgG seems to be the antibody subclass mostly responsible as

increases in anti-aGal IgG of 100-300-fold have been documented after experimental

pig organ and bone marrow transplantation in humans and nonhuman primates (31,

15

Section 1 - Chapter 1

32). These induced antibodies initiate AHXR by mechanisms that appear to be

independent of complement, although complement fractions may playa role (17,30).

Cells, such as macrophages, NK cells, and monocytes are present in the rejected

xenografts at the time of AHXR, suggesting that they may be important to the

rejection process (16, 33, 34). Macrophages, in particular, play an important role in

the production, mobilization, activation, and regulation of immune effector cells.

They participate in the activation of B and T cells. They process and present antigens,

secrete various cytokines and chemokines, and phagocytose apoptotic and necrotic

cells as well as various pathogens. It is not clear, however, if these cells, as well as

NK cells, playa direct role in mediating or effecting AHXR, or if they migrate to the

graft as a result of the presence of antibody.

\Vhatever the mechanism of AHXR, there is growing evidence that it is associated

with a state of disseminated intravascular coagulation in the recipient, which may

develop before histopathological evidence of AHXR is advanced (32, 35). This

phenomenon will de discussed in more detail in chapter 7.

Immunological Events following Antibody-Mediated Rejection

Since it has to date not been possible to routinely avert AHXR, discussion of the

immunological processes following AHXR is mostly hypothetical. However, in vitro

and in vivo studies involving discordant xenogeneic cells or grafts have shown that

pig tissues are also rejected by cellular (antibody-independent) mechanisms (36, 37,

38, 39, 40). The transplantation of pig pancreatic islets may be a useful model in that

these are transplanted in the absence of a vascular endotheliwn, and therefore do not

express aGal. Nevertheless, porcine islets are rejected by a cellular mechanism in

which macrophages and NK cells playa significant role. Although this model cannot

be fully extrapolated to the transplantation of a vascularized organ, it seems likely

that, if AHXR is prevented, a cellular form of rejection will take place in the

vascularized organ. Furthermore, chronic rejection, e.g. graft atherosclerosis, is likely

to develop early.

16

Introduction

1.3 MODULATION OF THE XENOGENEIC IMMUNE RESPONSE

HAR can be prevented by i) depletion/inhibition of anti-aGal, ii) depletion or

inhibition of components of complement, or iii) the use of organs from pigs transgenic

for human complement regulatory proteins. To date, it has not been possible to

prevent AHXR in the pig-to-nonhurnan primate model. Several methods, largely

aimed at depletion/inhibition of anti-aGal combined with prevention of production of

induced antibody, are currently being studied. Since non-specific cells, such as

macrophages and NK cells, appear to be important in the pathogenesis of AHXR.

agents targeting these cells may also be essential in preventing AHXR.

Present progress with these various therapeutic modalities are presented and discussed

in this chapter. The necessity for, and current methods of, depletion and suppression

of production of anti-aGal antibodies will be discussed in Chapters 3.

Depletion or Inhibition of Complement

In the 1960s, cobra venom was important m elucidating the activation of the

complement pathway (41). Cobra venom factor (CVF) was purified and was found to

activate the complement system as a functional analogue of C3b (42, 43). Similar to

C3b, CVF can bind to factor B, but is approximately 5 times more stable than C3bBb

and is resistant to decay acceleration and proteolytic inactivation (44). The

administration of CVF therefore leads to continuous complement activation, resulting

in depletion of one or more complement components. Complement depletion by CVF

can prolong discordant xenograft survival from minutes to days, but AHXR

eventually develops by complement-independent mechanisms (14, 45, 46, 47).

Inhibition of complement activation can be achieved successfully by administering

human soluble complement receptor 1 (sCR1). This leads to acceleration of the

proteolytic cleavage of C3!C5-convertases, thus preventing development of MAC and

proinflammatory cytokines. sCRI does not activate and deplete complement

components and may therefore be less toxic than CVF. Administered alone, sCRl can

also prolong discordant cardiac xenograft survival in the guinea pig-to-rat model to 32

17


hours (48) and in the pig-to-primate model to 7 days (49). When combined with CPP,

cyc1osporine and steroids, survival of pig cardiac xenografts was further prolonged to

a maximum of 6 weeks (50, 51).

Genetic Engineering of the Organ-Source Pig

To date, it has proven rather difficult to maintain depletion of anti-aGal antibodies in

the recipient primate. A different approach would be to prevent expression of the

aGal epitope on the vascular endothelial cells of the pig by genetic manipulation. It is

currently not possible to create an aGal-knockout pig (i.e., to disrupt the

al,3galactosyl-transferase gene (aGT) by homologous recombination, thereby

preventing aGal expression) because pig embryonic stem cells are not yet available.

Furthermore, studies in aGal-knockout mice indicate that the absence of aGal may

induce expression of underlying 'cryptic' oliogosaccharide epitopes to which humans

may have antibodies (52). However, several advances have been made in the field of

genetic engineering and are discussed below.

Transgenesis for human complement regulatory proteins

Since complement activation in humans is regulated by membrane bound complement

regulatory proteins, such as CD46 (membrane cofactor protein, MCP), CD55 (decay

accelerating factor, DAF), and CD59, inducing expression of these human proteins in

pigs to inhibit complement activation seems to be a logical approach. Complement

regulatory proteins are largely species-specific, i.e., complement regulatory proteins

expressed on porcine tissue do not modulate human complement and vice-versa. The

creation of pigs transgenic for human complement regulatory proteins therefore seems

desirable. \Vhite's group in C(lmbridge has successfully created pigs transgenic for

hDAF. 'When organs from these pigs are transplanted into nonhuman primates, they

are protected from HAR and, in association with intensive immunosuppressive

therapy, graft survival of up to 99 days has been reported (White, personal

communication). Median graft survival of life-supporting pig kidneys transplanted

into cynomolgus monkeys is approximately 30 days and of pig hearts transplanted

orthotopically into baboons about 15 days (18,19). Other groups have created pigs

18

Introduction

transgenic for more than one human complement regulatory protein but with less

prolonged graft survival (53), probably due to differences in the immunosuppressive

protocol administered.

Competitive glycosylation

As it is not yet possible to create an aGal-knockout pig, alternatives have been

proposed to decrease the aGal expression on donor pig organs. The introduction of a

gene that would compete with aGT for its substrate, N-acetyllactosamine, is one such

approach (54) (Figure 1). In vitro studies by Sandrin et a1. (55, 56) in COS cells

demonstrated that competition takes place between aGT and a 1 ,2fucosyltransferase

(aFT) in the Golgi apparatus for this substrate, and that aFT takes precedence,

thereby resulting in a cell that expresses more H blood group antigen than Gal. COS

cells simultaneously transfected with cDNA clones encoding for either aGT or aFT

showed greater expression of the uFT-product (the H epitape) than the uGT-product

Ct 1 ,3G alactosy Itra 0 sferase

Gal~I·4GlcNAc-R -----fIt-- Galul-3Galf}I-4GlcNAc-R

al,2Fucosyitransferase ~! ! ~ aGalactosidase

Gal~I-4GIcNA~_R Galf}I-4GIcNAc-R

I 0.1-2

Figure L

Natura! biosynthetic pathway for synthesis of the Gal epitope (Galal-3Gal), and methods by which this

can be modified by transgenic techniques. Galactose is added to the N-acetyllactosamine (GaI131-

4GlcNAc) substrate by the al,3 galactosyltransferase enzyme to fonn Gala J -3Ga1. Gall31-4G1cNAc

can also fonn the substrate for the H (0) histo-blood group epitope when the gene for the a 1,2

fucosyltransferase enzyme is transgenically introduced. FurthemlOre, cleavage of Gala 1-3Gal occurs

when the gene for the a-galactosidase enzyme is introduced. Modification of the natural pathway has

been demonstrated in cells in culture and in aGal-knockout (aGal-negative) mice by transgenic

techniques, but has not yet been successfully achieved in pigs. (Modified fTom Sandrin MS, et al.

(123)).

19

Section 1 - Chapter I

(aGal) (56). Mice transgenic for aFT demonstrated a major decrease in aGal

expression and reduction of reactivity of these cells when challenged with human

serum (57). More recently, evidence has been presented that high-level expression of

the H antigen on porcine cells reduces human monocyte adhesion and activation (58).

It would be necessary, however, to ensure that all the aGal epitopes are replaced by

the H epitopes, or the transplanted organ may still be susceptible to HAR or AHXR.

It has therefore been proposed to add another gene, namely a-galactosidase, to the

donor pig in addition to that for aFT (59). aGalactosidase removes terminal aGal

rather than adding it, and would ensure that those aGal epitopes that are not

competitively replaced with H epitopes by aFT are removed by a-galactosidase.

Sandrin's group has provided data demonstrating a complete absence of aGal

expression in cell cultures containing the genes for both aFT and a-galactosidase

(60).

Nuclear transfer

Our knowledge of and ability to genetically manipulate embryonic and adult cells of

large mammals has greatly increased in the last few years (61). The recent successful

cloning of pigs by PPL Therapeutics (62) holds considerable potential for the field of

xenotransplantation. Although the current cloned pigs are genetically unmodified, this

technology may allow the cloning of pigs that are genetically altered to render their

organs less susceptible to rejection by humans recipients. The technique by which

PPL Therapeutics achieved this success, nuclear transfer, had already led to the

cloning of the umuodified sheep, Dolly, in 1996 (61) and the genetically-modified

sheep, Polly, in 1997 (63). In Polly, the gene encoding the human clotting factor IX

had been inserted.

Nuclear transfer entails the in vitro removal of the nucleus from an unfertilized egg,

and its replacement with a nucleus removed from another cell of interest. This latter

cell can be a normal, unmodified embryonic, foetal or adult cell or a cell in which the

genome has been modified. The newly-created egg is initially cultured for a few days

20

Introduction

to allow cell division to begin, and the developing embryo is then implanted into the

uterus of a surrogate mother. The offspring is a (modified or umnodified) clone of the

pig that donated the original nuclear materiaL The potential benefit for

xenotransplantation lies in the possibility of deleting or inserting genes of interest in

the genome before its transfer, and thus create a genetically-modified pig. Advantages

over other methods of genetic modification are that (i) it does not require embryonic

stem cells (that to date remain unavailable in pigs), (ii) it allows for the assessment of

success of the genetic manipulation at an early (cellular) stage, thus negating the

costly wait until the offspring is born (as is the case with current transgenic

techniques), and (iii) it has the potential to produce an almost instantaneous herd of

identical modified swine.

In xenotransplantation, modification of the genome and nuclear transfer could, for

example, enable the production of aGal-knockout pigs by deleting the gene for

al,3galactosyltransferase in the npdeus before transfer, theoretically rendering the

resulting pig's organs resistant to a human recipient's antibody-mediated immune

response as discussed above. It remains uncertain, however, whether an aGal

knockout pig will be viable as so much aGal is present in the pig that some

authorities have questioned whether it may be essential to sustain life (64).

Alternatively or additionally, certain protective genes might be inserted, e.g., those for

one or more complement regulatory proteins, those known to be associated with the

development of accommodation (65), as discussed below, or those that might promote

thromboregulation (66), Physiologic barriers could also be surmounted, e.g., by the

introduction of a gene responsible for the production of a human protein, enzyme or

honnone (whenever the porcine equivalent is found not to function satisfactorily in

primates).

The technique of nuclear transfer will need considerable refinement and testing before

its implementation into clinical xenotransplantation, but its recent successful

attainment in pigs brings optimism for the future.

21

Section! - Chapter 1

Accommodation

Under certain circumstances, ABO-incompatible allografts or allografts in HLA

sensitized recipients are able to survive despite the presence of circulating antibodies

directed against detenninants on the grafted organ (67). This process has been tenned

accommodation, and can be summarized as the absence of antibody-mediated

rejection of a primary vascularized organ despite the presence of circulating

antibodies that are potentially reactive with antigens on the vascular endothelium of

that graft (68). The phenomenon has not yet been documented to conclusively occur

in Gal-incompatible large animal models. Possible explanations for the phenomenon

of accommodation have been proposed by Bach et al. (68) and are briefly reviewed.

Firstly, the returning antibodies, i.e. the antibodies that are induced post-transplant

following pre-transplant depletion, may be different in isotype, affinity, and/or

specificity, and are thus unable to initiate rejection. Secondly, the surface antigens on

the vascular endothelium may show subtle changes during the absence of natural

antibodies, thus preventing recognition by the returning, induced antibodies. Finally,

during the return of antibodies, the endothelial cells may adapt and either respond

differently to these antibodies or become 'desensitized'. It has been proposed that,

during accommodation, 'beneficial' genes are upregulated and 'detrimental' genes are

downregulated (65).

Induction of Tolerance

Many investigators believe that the ideal method for avoiding both HAR and AHXR

is to induce B cell tolerance. Additionally, if T cell tolerance were also induced, the

subsequent cellular response would be avoided. This would prevent the complications

associated with long-tenn pharmacological immunosuppressive therapy. Since

tolerance can be defined as a state of permanent specific unresponsiveness to donor

antigens (but not to other antigens) by the recipient in the absence of maintenance

immunosuppressive therapy, immune responses to pathogens would be nonna!. Sachs

and Sykes have developed tolerance in small and large animal allotransplantation

models (31, 32, 69, 70).

22

Introduction

Molecular chimerism

One approach to the induction of tolerance is by gene therapy in an attempt to induce

what has been termed molecular chimerism. For example, B cell tolerance might be

achieved if the primate recipient could be induced to express the Gal epitope on its

tissues, which might lead to the suppression of production of anti-Gal antibody.

Autologous transplantation of bone marrow from aGal-knockout mice transduced ex

vivo with the gene for agalactosyltransferase (aGT, the enzyme that leads to the

production of the Gal epitopes) resulted in suppression of production of anti-a Gal and

the achievement of B cell tolerance to aGal (71). Preliminary studies in baboons,

however, demonstrated only transient expression of aGal following the infusion of

transduced autologous bone marrow cells. The transduction efficiency of baboon bone

marrow ceils is currently being optimized with the use of improved vectors and

culture parameters.

T cell tolerance might be achieved by the introduction into the recipient of a gene

encoding a swine MHC (SLA) class II antigen. The presence of a donor-specific class

II antigen in the recipient (following gene transduction of bone marrow cells) has

been demonstrated to lead to tolerance to a kidney allograft in miniature swine (72),

and its importance has been tested in the pig-to-baboon model (73). Ex vivo

transduction of baboon bone marrow cells with an SLA class II gene of a specific

MHC-inbred miniature swine genotype was performed. Autologous transplantation of

these transduced cells led to detection of the transgene in blood and bone marrow, but

the transcription was transient. Subsequent pig skin or organ grafts (from a pig

matched to the trans gene ) were rejected within 8 to 22 days from an antibody

mediated mechanism. In contrast to control baboons, however, the induction of IgG

against non-a Gal antigens was prevented, suggesting prevention of a T cell response.

Thymic transplantation

Zhao et al. demonstrated that the transplantation of fetal pig thymus and liver tissue

(as a source of hematopoietic cells) under the kidney capsule of thymectomized and T

and NK cell-depleted mouse recipients can induce donor-specific tolerance and

23

Section / - Chapter I

restore immune competence (74). Furthermore, when donor-matched pig skin grafts

were transplanted to these mice, permanent graft survival was achieved, whereas

allogeneic skin grafts rejected within 26 days (75). These studies were expanded by

Wu et a!., who demonstrated in vitro unresponsiveness fo llowing fetal porcine thymic

tissue grafting in thymectomized, T cell -depleted baboons (76). Further studies are

underway in this model.

Figu re 2.

Microscopic appearance (I OOx) of pig thym ic tissue 3 months after transplantation under the capsule of

an autologous kidney. Kidney tissue can be seen at the right margin of the figure; nOle the complete

reorganization of the thymic tissue, which allowed normal thymopoiesis.

Our center is also involved with the induction of T cell tolerance by the

transplantation of a vascularized thymic graft from the donor. Yamada et a!. have

demonstrated in our MHC-inbred herd of pigs that autologous thymic ti ssue, when

transplanted under the renal capsule, becomes vascularized, regenerates, and functions

normally (77) (Figure 2). When these "thymo-kidney" composite grafts are

transplanted across a full y-mismatched allogeneic barrier, in a T cell depleted and

thymectomized recipient, they are able to induce T cell tolerance (78). If anti-aGal

could be successfully depleted for a limited period of time, the transplantation of a pig

thymokidney could potentially induce T cell tolerance in the pig-to-primate model.

The problem of providing an anti-Gal-free environment that would allow T cell

to lerance to develop, however, remains unresolved at the present time.

24

Introduction

Figure 3.

Thymic irradiation Whole body irradiation +

AntiMthymocyte globulin Splenectomy + +

Extracorporeal immunoadsorption

-B -7 -6 -s -4 -3 -2 -1 0

Days

2 3

Mycophenolate mofetil +/M Cyclosporine ... ---------4~~

Cobra venom factor

~ AntiMCDl54 mAb

Porcine Ilr3 and Porcine Stem cell factor

Standard nonMmyeloab lative reg imen aimed at inducing immuno logical tol erance. The induction

therapy fo r baboons cons ists ofa splenectomy on day -8, followed by whole body irradiation 150 cGy

x2 on days -6 and - 5 to fac il itate engraftment of porcine ce lls. T cell depletion is achieved with

anti thymocyte globulin on days - 3, -2, and - I and thymic irradiation 700 cGy on day -I.

Extracorporeal immunoadsorption of antiMuGal Ab is perfonned on days -2, - I, and O. The

maintenance therapy consists of mycophenolate mofetil at 110 mg/kglday by continuous iv infusion

from days -6 to 21, complement depletion with cobra venom factor from days - I to 28, costimulatory

bl ockade with antiMCD 154 mAb from days 0 to 14 at 20 mg/kg on alternate days, either with or without

cyclosporine at 15 mg/kglday by continuous iv infusion. PBPC are transplanted on days 0, I, and 2 (not

shown). Porcine growth factors f iLM) 400 JlglkgJday and stem cell factor 2000 Jlg/kg/day) are

administered from days 0 to 21 by continuous iv infusion to promote engraftment of porcine cells.

Mixed hematopoietic cell chimerism

Another approach to inducing tolerance to a transplanted xenogeneic organ would be

to first induce mixed hematopoietic cell chimerism between pig and primate (32). To

induce this state in the pigMtoMprimate model, a combination of (at least temporary) (i)

depletion or inhibi tion of anti-aGal, (ii ) depletion or inhibition of complement, (i ii) T

cell depletion (and/or costimulatory blockade), and (iv) pig hematopoietic cell

engraftment, would appear to be necessary. Our laboratory is involved in studies

aimed at inducing mixed hematopoietic chimerism and thus a state of both B and T

cell tolerance in this model. We infuse large quantities of cytokine-mobilized

peripheral blood progenitor cells (approximately 2 - 4 x 10'0 cellslkg), obtained from

25


our MHC-inbred herd of miniature swine, into a pre-conditioned recipient baboon.

Our current conditioning regimen consists of splenectomy, \VBI, thymic irradiation,

anti-thymocyte globulin, MMF, CyA, CVF, anti-CDl54 mAb, and multiple EIAs

(Figure 3).

20 18

'" 16

E 14

~ 12

• 10 E 8 :2 u 6

4 2 0

Figure 4.

0 5

1ft Pi~ PBPC

-Pan Pig

~CD9

.. ().- Pig MAC

10 15 20 25 30 35

DAYS

Pig celt chimerism detected by flow cytometry in the blood of a baboon treated with our

immunomodulatory regimen (see Figure 3) . Porcine PBPC were infused on days 0 - 2, during which

up to 16% of the white blood cells in the baboon were of pig origin. From days 5 through 15 no pig

cells could be detected, but on days 16 through 21 a pig monocyte population (stained with markers for

pig (pan pig), pig CD9 (platelets, early B cells, activated T cells, and basophils), and pig MAC

(monocytes and granulocytes)) could again be detected, indicative of transient pig cell engraftment in

the baboon.

With this regimen, we are able to detect porcine cells by flow cytometry until day 6

following infusion, and by polymerase chain reaction consistently through day 30 and

intermittently up to 140 days. Although engraftment of porcine cells, measurable by

flow cytometry, has been definitively documented only twice, a state of donor

specific hyporesponsiveness, detected by mixed lymphocyte reaction from day 40 -

80 (Figure 3), has been observed in several cases, indicating that the induction of

tolerance through mixed hematopoietic chimerism seems feasible if anti-Gal can be

removed for a sufficient period of time.

26

introduction

In our experience, the experiments aimed at inducing immunological tolerance

through mixed chimerism, have been associated with severe thrombotic

complications. Most baboons that underwent our tolerance inducing protocols

developed a thrombotic microangiopathy, resulting in massive bleeding complications

and even death of several baboons. Although these thrombotic complications appear

to be quite different than the disseminated intravascular coagulation that is observed

after solid organ pig-to-baboon xenotransplantation, it is possible that the mechanisms

that playa role in the etiology of these events may be similar.

1.4 AIMS OF THIS DISSERTATION

Xenotransplantation has the potential to change transplantation medicine as it is

currently known. No longer will patients with organ failure need to wait weeks,

months or years for an organ as a limitless number of animal organs will be readily

available. The surgical procedure can be planned electively, during normal working

hours, while the patient is still in reasonable health. The use of animals as the source

of organs also presents us with new opportunities to manipulate and/or modify the

"donor" using techniques such as genetic engineering and nuclear transfer.

Manipulation of the donor has not been possible in allotransplantation.

The rejection processes that take place following th~ transplantation of a vascularized

organ from a pig to a primate present formidable barriers. Much progress has been

made in the last decade in elucidating the pathophysiology of these phenomena.

Although major immunological hurdles (such as the prevention of HAR) have been

overcome, many others remain. It is unlikely that any of the therapeutic modalities

discussed in this chapter will be successful in preventing rejection when used alone. A

combination of therapies will almost certainly be necessary, and we believe that the

successful induction of immunological tolerance will greatly facilitate clinical

implementation of xenotransplantation.

27

Section 1- Chapter I

The aims of this dissertation are to discuss the current status of the induction of

immunological tolerance in pig-to-baboon xenotransplantation through mixed

hematopoietic chimerism, and to investigate some of the hurdles and complications

associated with this approach. The author wishes to stress that only immunological

aspects of xenotransplantation are presented, and that no attempts will be made to

discuss the equally important issues of infectious hazards, ethical considerations, and

physiological barriers that are inherent to xenotransplantation, as these latter items fall

beyond the scope of this dissertation.

The results obtained from our attempts to induce mixed hematopoietic chimerism by

depleting, or inhibiting the function of, macrophages in the pig-to-baboon model are

presented in this thesis (Section II, Chapter 2).

In order to achieve mixed hematopoietic chimerism, anti-aGal depletion, and

maintenance of depletion for a significant period of time, possibly several weeks, is

required. We have summarized current and novel methods of depletion or suppression

of production of anti-pig antibodies in pig-to-primate xenotransplantation (Chapter

3). Furthermore, the effect of depletion of B cells by specific immunotherapy on anti

aGal production in baboons is presented (Chapter 4).

The potentially lethal thrombotic microangiopathy that is associated with the

transplantation of large quantities of porcine hematopoietic cells is discussed in

Section III, Chapter 5, and various etiology is presented. The direct effect of porcine

cells on baboon platelet aggregation is investigated (Chapter 6), and potential

beneficial or detrimental aspects of our conditioning regimen aimed at inducing

mixed chimerism are examined (Chapter 7). Finally, several agents that may prevent

platelet aggregation in baboons are examined (Chapter 8).

These data are summarized in Section IV, Chapter 9.

28

Introduction

REFERENCES

1 Harper AM, Rosendale JD. The UNOS OPTN waiting list and donor registry. Clin Transp11997: 61 2 Delmonico FL, Harmon WE, Lorber MI, et al. A new allocation plan for renal transplantation.

Transplantation 1999; 67:303 3 Aaronson KD, Mancini OM. Mortality remains high for outpatient transplant candidates with

prolonged (>6 months) waiting list time. J Am Coil Cardio11999; 33(5): 1189 4 Cooper DKC. Xenografting: how great is the clinical need? Xeno 1993; 1: 25 5 Lambrigts 0, Sachs DH, Cooper DKC. Discordant organ xenotransplantation in primates. World

experience and current status. Tranplantation 1998; 66: 547 6 Bailey LL, Gundry SR. Survival following orthotopic cardiac xenotransplantation between juvenile

baboon recipients and concordant and discordant donor species: foundation for clinical trials. World J Surg 1997; 21 :943

7 Perper RJ, Najarian JS. Experimental renal heterotransplantation. I. In widely divergent species. Transplantation 1966; 4: 377

8 Cooper DKC, Human PA, Lexer G, et al. Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the baboon. J Heart Lung Transplant 1988; 7: 238

9 Good AH, Cooper DKC, Malcolm AJ, et al. Identification of carbohydrate structures that bind human anti-porcine antibodies: implications for discordant xenografting in humans. Transplant Proc 1992; 24: 559

10 Platt JL, Fischel RJ, Matas AJ, et al. Immunopathology of hyperacute xenograft rejection in a swine-to-primate model. Transplantation 1991; 52: 514

II Rose AG, Cooper DKC, Human PA, et al. Histopathology of hyperacute rejection of the heart: experimental and clinical observations in allografts and xenografts. J Heart Lung Transplant 1991; 10: 223

12 Rose AG, Cooper OK. A histopathologic grading system of hyperacute (humoral, antibodymediated) cardiac xenograft and allograft rejection. J Heart Lung Transplant 1996; 15:804

13 Alexandre GPJ, Gianello P, Latinne 0, et al. Plasmapheresis and splenectomy in experimental renal xenotransplantation. In: Hardy MA (ed). Xenograft 25. New York, Elsevier, 1989. p 28.

14 Leventhal JR, Dalmasso AP, Cromwell JW, et al. Prolongation of cardiac xenograft survival by depletion of complement. Transplantation. 1993; 55: 857

15 Cozzi E, White DJG. The generation of transgenic pigs as potential organ donors for humans. Nat Med 1995; 1: 964

16 Fryer JP, Leventhal JR, Dalmasso AP, et al. Cellular rejection in discordant xenografts when hyperacute rejection is prevented: analysis using adoptive and passive transfer. Transpllmmunol 1994; 2:87

17 Bach FH, Robson SC, Winkler H, et al. Barriers to xenotransplantation. Nat Med 1995; 1: 869 18 Schmoeckel M, Bhatti FN, Zaidi A et al. Orthotopic heart transplantation in a transgenic pig-to

primate model. Transplantation 1998; 65: 1570 19 Zaidi A, Schmoeckel M. Bhatti F, et al. Life-supporting pig-to-primate renal xenotransplantation

using genetically modified donors. Transplantation 1998; 65: 1584 20 Cooper DKC, Ye Y, Rolf LL Jr, et al. The pig as a potential organ donor for man. In: Cooper DKC,

Kemp E, Reemtsma K, et al. (eds.). Xenotranplantation. Heidelberg, Springer 1991. p481 21 Sachs DH. The pig as a potential xenograft donor. Vet Imm Immunopath 1994; 43: 185 22 Galili U, Shohet SB, Kobrin E, et al. Man, apes, and Old World monkeys differ from other

mammals in the expression of a-galactosyl epitopes on nucleated cells. J Bioi Chern 1988; 263: 17755

23 Oriol R, Ye Y, Koren E, et al. Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation. Transplantation 1993; 56: 1433

24 Galili U, Rachmilewitz EA, Peleg A, et al. A unique natural human IgG antibody with anti-ugalactosyl specificity. J Exp Med 1984; 260: 1519

25 Cooper DKC, Good AH, Koren E, et al. Identification of a-galatosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies: relevance to discordant xenografting in man. Transplant Immunol 1993; 1: 198

26 Cooper DKC, Koren E, Oriol R. Oligosaccharides and discordant xenotransplantation. Immunol

29

Section 1- Chapter J

Rev 1994; 141: 31 27 Kujundzic M, Koren E, Neethling FA, et al. Variability of anti-aGal antibodies in human serum and

their relation to serum cytotoxicity against pig cells. Xenotransplantation 1994; I: 58 28 McMorrow 1M, eomrack CA, Nazarey PP, et al. Relationship between ABO blood group and levels

of Gal alpha,3galactose-reactive human immunoglobulin G. Transplantation 1997; 64: 546 29 Sandrin MS, Vaughan HA, Dabkowski PL, et al. Anti-pig IgM antibodies in human serum react

predominantly with Gala(1-3)Gal epitopes. Proc Nat! Acad Sci USA 1993; 90: 11391 30 Galili U. Anti-agalactosy1 (anti-Gal) antibody damage beyond hyperacute rejection. In: Cooper

DKC, Kemp E, Platt JL, et al. (eds). Xenotransplantation, 2nd edition. Heidelberg, Springer 1997. P 95

31 Kozlowski T, Monroy R, Xu, Y, et al. Anti-Galal-3Gal antibody response to porcine bone marrow in unmodified baboons and baboons conditioned for tolerance induction. Transplantation 1998; 66: 176

32 Kozlowski T, Shimuzu A, Lambrigts D, et al. Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption. Transplantation 1999; 67: 18

33 eandinas 0, Belliveau S, Koyamada N, et a1. T cell independence of macrophage and natural killer cell infiltration, cytokine production, and endothelial activation during delayed xenograft rejection. Transplantation 1996; 62:1920

34 Marquet RL, van Overdam K, Boudesteijn EA, et a1. ImmunobioJogy of delayed xenograft rejection. Transplant Proc 1997; 29:955

35 lerino FL, Kozlowski T, Siegel 18, et al. Disseminated intravascular coagulation in association with the delayed rejection of pig-to-baboon renal xenografts. Transplantation 1998; 66: 1439 .

36 Moses RD, Auchinc10ss H. Mechanism of cellular xenograft rejection. In: Cooper DKC, Kemp E, Platt JL, et al. (ed.), Xenotransplantation 2nd edition. Heidelberg, Springer 1997. p 140

37 Vallee I, Guillaumin JM, Thibault G, et al. Human T lymphocyte proliferative response to resting porcine endothelial cells results from an HLA-restricted, IL-J 0 sensitive, indirect presentation pathway but also depends on endothelial-specific costimulatory factors. J Immunol 1998; 161: 1652

38 Simeonovic eJ, Townsend MJ, Morris eF, et al. Immune mechanisms associated with the rejection of fetal murine proislets aUografts and pig proislet xenografts: comparison of intragraft cytokine mRNA profiles. Transplantation 1999; 67:963

39 Soderlund J, Wennberg L, Castanos-Velez E, et al. Fetal porcine islet-like cell clusters transplanted to cynomolgus monkeys: an immunohistochemical study. Transplantation 1999; 67:784

40 Siebers U, Horcher A, Brandhorst H, et al. Analysis of the cellular reaction towards microencapsulated xenogeneic islets after intraperitoneal transplantation. J Mol Med 1999; 77:215

41 MuUer Eberhard HJ, Nilsson UR, Dalmasso AP, et al. A molecular concept of immune cytolysis. Arch Path 1966; 82:205

42 Alper CA, Balatovich D. Cobra venom factor: evidence for its being altered cobra C3 (the third component of complement). Science 1976; 191: 1275

43 Vogel CW, Smith CA, Muller Eberhard HJ. Cobra venom factor: structural homology with the third component of human complement. J Immunol 1984; 133;3235

44 Lachmann PJ, Halbwachs L. The influence ofC3b inactivator (KAF) concentration on the ability of serum to support complement activation. Clin Exp Immunol 1975; 21: I 09

45 Adachi H, Rosengard BR, Hutchins GM, et al. Effects of cyclosporine, aspirin, and cobra venom factor on discordant cardiac xenograft survival in rats. Tranplant Proc 1987; 19: 1145- 1148

46 Scheringa M, Schraa EO, Bouwman E, et al. Prolongation of survival of guinea pig heart grafts in cobra venom factor~treated rats by splenectomy. No additional effect of cyclosporine. Transplantation 1995; 60: 1350

47 Kobayashi T, Taniguchi S, Neethling FA, et al. Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy. Transplantation 1997; 64: 1255

48 Candinas D, Lesnikoski BA, Robson SC, et al. Effect of repetitive high-dose treatment with soluble complement receptor type I and cobra venom factor on discordant xenograft survival. Transplantataion 1996; 62:336

49 Pruitt SK, Bollinger RR, Collins BH, et al. Effect of continuous complement inhibition using soluble complement receptor type 1 on survival of pig-to-primate cardiac xenografts. Transplantation 1997; 63:900

30

Introduction

50 Davis EA, Pruitt SK, Greene PS, et al. Inhibition of complement, evoked antibody, and cellular response prevents rejection of pig-to-primate cardiac xenografis. Transplantation 1996; 62: 1018

51 Marsh He Jr, Ryan US. Therapeutic effect of soluble complement receptor type I in xenotransplantation. In: Cooper DKC, Kemp E, et al. eds. Xenotransplantation, 2nd edition. Heidelberg, Springer 1997. p 437

52 Shinkel T A, Chen CG, Salvaris E, et al. Changes in cell surface glycosylation in alpha 1,3-galactosyltransferase knockout and alpha 1,2-fucosyltransferase transgenic mice. Transplantation 1997; 64; 197

53 Lin SS, Weidner BC, Byrne GW, et al. The role of antibodies in acute vascular rejection of pig-toprimate cardiac transplants. J Clin Invest 1998; 101:1745

54 Cooper DKC, Koren E, Oriol R. Genetically engineerd pigs (Letter). The Lancet 1993; 342:682 55 Sandrin MS, Fodor WL, Mouhtouris E, et al. Enzymatic remodelling of the carbohydrate surface of

a xenogenic cell substantially reduces human antibody binding and complement-mediated cytolysis. Nature Med 1995; 1:1261

56 Sandrin MS, Cohney S, Osman N. et al. Overcoming the anti-Galal-3Gal reaction to avoid hyperacute rejection: molecular genetic approaches. In: Cooper DKC, Kemp E, Platt JL, et at. (eds.) Xenotransplantation, 2nd edition. Heidelberg, Springer 1997. p683

57 Chen CG, Salvaris EJ, Romanella M, et al. Transgenic expression of human alphal,2-fucosyltransferase (H-transferase) prolongs mouse heart survival in an ex vivo model of xenograft rejection. Transplantation 1998; 65:832

58 Kwiatkowski P, Artrip JH, Edwards NM, et al. High-level porcine endothelial cell expression of alpha(l ,2)-fucosyltransferase reduces human monocyte adhesion and activation. Transplantation 1999; 67;219

59 Cooper DKC, Koren E, Oriol R. Manipulation of the anti-aGal antibody-aGal epitope system in experimental discordant xenotransplantation.Xenotransplantation 1996; 3: 1 02

60 Osman N, McKenzie IF, Ostenried K, et al. Combined transgenic expression of alpha-galactosidase and alpha 1 ,2-fucosyltransferase leads to optimal reduction in the major xenoepitope GaJaJpha (1,3) Gal. Proc Nat] Acad Sci USA 1997; 94: 14677

61 Wilmut L Schnieke AE, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells. Nature 1997; 385:810

62 Dobson R. Cloning of pigs bring xenotransplants closer. BMJ 2000; 320: 826 63 Schneike AE, Kind AJ, Ritchie WA, et al. Human factor IX transgenic sheep produced by transfer

ofnucJei from fetal fibroblasts. Science 1997; 278: 2130 64 Tanemura M, Maruyama S, Galili U. Differential expression of alpha-Gal epitopes (Galalphal-

3Galbetal-4G1cNAc-R) on pig and mouse organs. Transplantation 2000; 69: 187 65 Bach FH, Ferran C, Hechenleitner P, et al. Accommodation of vascularized xenografts: expression

of "protective genes" by donor endothelial cells in a host Th2 cytokine environment. Nat Med. 1997; 3; 196

66 Chen D, Riesbeck K, McVey JH, et a1. Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins. Transplantation 1999; 68: 832

67 Chopek MW, Simmons RL, Piatt JL. ABO incompatible renal tTansplantation: initial immunopathologic evaluation. Transplant Proc 1987; 19: 4553

68 Bach FH, Platt JL, Cooper DKC. Accommodation - the role ofnatura\ antibody and complement in discordant xenograft rejection. In: Cooper DKC, Kemp E, Reemtsma K, et aL (eds). Xenotransplantation. Heidelberg, Springer 1991. p 81

69 Yang YG, DeGoma E, Ohdan H, et al. Tolerization of anti-Galal-3Gal natural antibody-forming B cells by induction of mixed chimerism. J Exp Med 1998; 187:1335-1342

70 Ohdan H, Yang YG, Shimizu A, et al. Mixed chimerism induced without lethal conditioning prevents T cell- and anti-Gal alpha 1,3Gal-mediated graft rejection. J Clin Invest 1999; 104:281

71 Bracy JL, Sachs DH, Iacomini J. Inhibition of xenoreactive natural antibody production by retroviral gene therapy. Science 1998; 281: 1845

72 Emery DW, Sablinski T, Shimada H, et al. Expression of an allogeneic MHC DRB transgene, through retroviral transduction of bone marrow, induces specific reduction of alloreactivity. Transplantation 1997; 64:1414

31

Section I - Chapter 1

73 lerino FL, Gojo S, BaneJjee PT, et al. Transfer of swine major histocompatibility complex class II genes into autologous bone marrow cells of baboons for the induction of tolerance across xenogeneic barriers. Transplantation 1999; 67: 1119

74 Zhao Y, Fishman lA, Sergio 11, et al. Immune restoration by fetal pig thymus grafts in T celldepleted, thymectomized mice. 1 Immunol1997; 158:1641

75 Zhao Y, Swenson K, Sergio lJ, et al. Skin graft tolerance across a discordant xenogeneic barrier. Nat Med 1996; 2:1211

76 Wu A, Esnaola NF, Yamada K, et al. Xenogeneic thymic transplantation in a pig-to-nonhuman primate model. Transplant Proc 1999; 31 :957

77 Yamada K, Shimizu A, lerino FL, et al. Thymic transplantation in miniature swine: l. Development and function of the "thymokidney". Transplantation 1999; 68:1684

78 Yamada K, Shimizu A, leriTIo FL, et al. Allogeneic thymokidney transplants induce stable tolerance in miniature swine. Transplant Proc 1999; 31: 1199

32

II Attempts to induce mixed hematopoietic chimerism in pig-to-baboon xenotransplantation

2 Depletion of macrophages in baboons delays the clearance of

mobilized pig lenkocytes

Adapted from: Murali Basker', Ian P.l. Alwayo*, Leo Buhler, David Harper, Sonny Abraham, Huw Kruger Gray, Michel Awwad, Julian Down, Robert Rieben, Mary E. White-Scharf, David H. Sachs, Aron Thall, and David K. C. Cooper. Clearance of mobilized porcine peripheral blood progenitor cells is delayed by depletion of the mononuclear phagocyte system in baboons. Transplantation 2001. In press . • Authors contributed equally.

Section IJ - Chapter 2

ABSTRACT

Introduction. Attempts to achieve immunological tolerance to porcine tissues in

nonhuman primates through establishment of mixed hematopoietic chimerism are

hindered by the rapid clearance of mobilized porcine leukocytes, containing

progenitor cells (pPBPC), from the circulation. Eighteen hours after infusing 1-2xl01o

pPBPC/kg into baboons that had been depleted of circulating anti-a Gal and

complement, these cells are almost undetectable by flow cytometry. The aim of the

present study Vias to identify mechanisms that contribute to rapid clearance of pPBPC

in the baboon. This was achieved by depleting, or blocking the Fe-receptors of, cells

of the mononuclear phagocyte system (MPS) using medronate liposomes (ML) or

intravenous immunoglobulin (lVIg), respectively.

:\Iethods. Baboons (Preliminary studies, n=4) were used in a dose-finding and

toxicity study to assess the effect of ML on macrophage depletion in vivo.

Furthennore, baboons (n=9) received a non-myeloablative conditioning regimen

(NMCR) aimed at inducing immunological tolerance, including splenectomy, whole

body irradiation (300 cGy) or cyclophosphamide (80 mg/kg), thymic irradiation (700

cGy), T cell depletion, complement depletion with cobra venom factor,

mycophenolate mofetil, anti-CD154L rnAb, and multiple extracorporeal

immunoadsorptions of anti-aGal antibodies. Group I (n~5) NMCR + pPBPC Tx.

Group 2 (n~2) NMCR + ML + pPBPC Tx. Group 3 (n~2) NMCR + IVlg + pPBPC

Tx. Detection of pig cells in the blood was assessed by FACS and PCR.

Results. Preliminary studies: ML effectively depleted macro phages from the

circulation in a dose-dependent manner. Group 1: On average, 14% pig cells were

detected 2 hours post-infusion of lxlO IO pPBPC/kg. After 18 hours, there were

generally less than 1.5% pig cells detectable. Group 2: Substantially higher levels of

pig cell chimerism (55-78%) were detected 2 hours post-infusion, even when a

smaller number (0.5-lxI010/kg) of pPBPC had been infused, and these levels were

better sustained 18 hours later (10-52%). Group 3: In one baboon, 4.4% pig cells were

detected 2 hours after infusion of lxl0 10 pPBPC/kg. After 18 hours, however, 7.4%

pig cells were detected. A second baboon died 2 hours after infusing 4xlO IO

pPBPC/kg, with a total white blood cell count of 90,000 of which 70% were pig cells.

36

Depletion of macro phages

No differences in microchimerism could be detected bet\.veen the groups as

determined by PCR.

Conclusions. This is the first study to report an efficient decrease of phagocytic

function by depletion of macrophages with ML in a large animal model. Depletion of

macrophages with ML led to initial higher chimerism and prolonged the survival of

circulating pig cells in baboons. Blockade of macrophage function with IVIg had a

more modest effect. Cells of the MPS. therefore, playa major role in clearing pPBPC

from the circulation in baboons. Depletion or blockade of the MPS may contribute

towards achieving mixed hematopoietic chimerism and induction of tolerance to a

discordant xenograft.

INTRODUCTION

The induction of stable mixed hematopoietic chimerism has led to transplantation

tolerance in several models of rodent (1,2,3.4), porcine (5). and primate (6.7)

allotransplantation, as well as in concordant rodent (8.9) and primate (10)

xenotransplantation. It has, however. not yet proven possible to achieve stable mixed

hematopoietic chimerism III the highly relevant pig-to-primate discordant

xenotransplantation modeL Our laboratory has developed a non-myeloablative

regimen (NMCR) aimed at inducing mixed hematopoietic chimerism in this model

that is based on protocols that have previously been successful in the allograft and

xenograft studies mentioned above. Large numbers of mobilized porcine peripheral

blood leukocytes, containing progenitors cells (pPBPC). are transplanted into baboons

pre-conditioned with a NMCR. Most pPBPC are, however. rapidly cleared from the

circulation of these baboons, as detem1ined by flow cytometric analysis of the

peripheral blood. As our NMCR includes therapies that deplete and block the function

of T cells, and deplete complement and circulating xenoreactive natural anti-Gala 1-

3Gal antibodies (anti-aGal Ab). we hypothesized that elements of the innate immune

system may be responsible for the rapid clearance ofpPBPC.

It has been well described that macrophages, as well as other cells of the phagocy1ic

reticulo-endothelial system, are associated with allo- and xenograft rejection

37


(11,12,13). Several groups have reported that depletion of these cells led to increased

graft survival (14, IS, 16, 17). Van Rooijen et al. have extensively studied the depletion

of macrophages using liposomes that contain the diphosphonate, clodronate

(18,19,20). These liposomes have distinct dimensions and properties, that render them

susceptible to phagocytosis by macrophages. Once phagocytosed, clodronate

interferes with the cell's metabolism and causes cell death. We have made similar

observations using liposomes containing medronate (ML), the parent compound of

clodronate, to which it has similar properties. We have previously demonstrated that

effective depletion of macrophages from the peripheral blood and other tissues is

achieved when ML are administered to mice (Cheng J, et al. manuscript in

preparation). To date, however, ML have not been studied in large animals, including

nonhuman primates.

Instead of depleting macrophages, an alternative would be to inhibit the fimction of

macrophages. Intravenous immunoglobulin (IVIg) is widely used in the treatment of

various autoimmune diseases, including idiopathic thrombocytopenic purpura (21).

Although the mechanism of action of IVlg has not been fully elucidated, it is thought

that one of the mechanisms through which IVIg may act is to bind to the Fc-ganuna

receptors of macrophages, thus inhibiting their ability to mediate antibody-dependent

cellular cytotoxicity (22). Furthermore, as IVlg may also inhibit complement activity

(23), and has been used in the treatment of humoral rejection in allotransplantation

(24,25), this action could be of additional benefit in xenotransplantation.

The present study was, therefore, designed to determine (i) whether ML are able to

deplete macrophages in baboons using a modified in vivo phagocytosis assay (26),

and (ii) whether the depletion (by ML) or blockade (by IVlg) of the cells of the MPS

delays the clearance of pPBPC following their transplantation in pre-conditioned

baboons.

38

Depletion of macrophages

MATERIAL & METHODS

Animals

Baboons (Papio anubis, n=13, Biomedical Resources Foundation, Houston, TX), of

known ABO blood group and weighing 9-15 kg, were used to assess the effect of ML

on macrophage depletion in vivo (n~4), or as recipients of pPBPC (n~9).

Massachusetts General Hospital MHC-inbred miniature swine (n=6, Charles River

Laboratories, Wilmington, MA), of blood group 0 and weighing 18-40 kg, served as

donors of pPBPC.

All experiments were performed in accordance with the Guide for the Care and Use of

Laboratory Animals prepared by the National Academy of Sciences and published by

the National Institutes of Health. Protocols were approved by the Massachusetts

General Hospital Subcommittee on Research Animal Care.

Surgical Procedures

Details of anesthesia, intravenous (iv) catheter placement in pigs and baboons, and

intra-arterial line placement, splenectomy, and extracorporeal immunoadsorption of

anti-aGal antibodies in baboons have been described previously (27,28).

Non-myeloablative Conditioning Regimen (NMCR)

This regimen consists of induction therapy with splenectomy, whole body irradiation

(150 cGy x2), thymic irradiation (700 cGy), anti-thymocyte globulin (50 mg/kg x3,

Atgarn, Upjohn, Kalamazoo, MI), and multiplc extracorporeal irnrnunoadsorptions of

anti-aGal antibodies. The maintenance therapy consists of treatment with anti-CD154

monoc1onal antibody (rnAb) +/- cyc1osporine (Sandirnrnune iv, 15 mg/kg/day by

continuous iv infusion, generously donated by Novartis Pharmaceuticals, East

Hanover, NJ), mycophenolate mofetil (Cellcept iv, 110 mg/kg/day by continuous iv

infusion, generously donated by Roche Laboratories, Nutley, NJ), and cobra venom

factor (Advanced Research Technologies, San Diego, CAl. Details of this regimen

can be found in Chapter 1, figure 3. All baboons received cefazolin, 500 mg iv daily,

as prophylaxis against infection and ranitidine, 12.5 mg iv twice daily (Zantac iv,

Glaxo-Wellcome, Research Triangle Park, NC).

39

Section /1- Chapter 2

Mobilization and Leukapheresis of pPBPC

Our methodology has been described previously (29). Briefly, pigs were treated with

recombinant hematopoietic growth factors before and during the period of collection

of pPBPC. Leukapheresis was carried out on days 5-9 and 12-16 following

commencement of growth factor mobilization using a Cobe Spectra apheresis

machine (Cobe, Lakewood, CO). Plasma was removed from the product by

centrifugation at 920g for 7 minutes. The collected leukocytes (pPBPC), containing

progenitor cells, were washed, frozen, and stored until transplantation.

Preparation and Transplantation of pPBPC

The pPBPC were thawed by immersion in a 37 - 40° C water bath with gentle shaking

for 1 - 2 minutes, washed twice with Hanks' balanced salt solution (ca1cium-,

magnesium-, and phenol red-free) (Mediatech, Herndon, VA), and resuspended in

Hanks' solution for immediate transplantation through a central systemic iv catheter

to the recipient baboon. Viability was assessed with trypan blue staining;

approximately 70-80% of pPBPC remained viable. The baboons received additional

therapy to prevent thrombotic complications associated with the transplantation of

pPBPC (30). This consisted of prostaeyelin, 20 ng/kglmin, and heparin, 10 U/kglhour

by continuous iv infusion, and methylprednisolone, 2 mg/kg iv tv.rice daily for 7 days,

tapered to daily for a further 7-21 days.

Preparation of Medronate Liposomes (ML)

This followed the method described by others (19). Briefly, 75mg phosphotidyl

choline (Avanti Polar Lipids, Alabaster, AL) and llmg cholesterol (Sigma, St. Louis,

MO) were dissolved in chloroform and mixed to form a film. After low vacuum

rotary evaporation at 37° C, the film was dispersed by gentle rotation in lOmI

phosphate buffered saline in which 2.5 mg medronate was dissolved. The resulting

liposomes were centrifuged twice at 100,000 g for 30 minutes to remove free, non

entrapped medronate. The ML were then resuspended in phosphate buffered saline

and ultrasonicated for 2 minutes before administration (Bransonic 2210, Branson

40

Depletion oJmacrophages

Ultrasonics Corporation, Danbury, CT). Blank liposomes were formed using the same

protocol with the omission of medronate.

Preparation of Fluorescent Latex Beads

Polystyrene latex beads, with a diameter of 2 fim (Polyscience, Warrington, PAl,

were impregnated with fluorescein isothiocyanate and added to tubes coated with

bovine serum albumin and rinsed with 70% ethanoL The resulting beads had

dimensions that made them particularly amenable to phagocytosis by macrophages.

They were stored in the dark in sterile normal saline at 4° C until use.

Preparation ofIntravenous Immunoglobulin (IVIg)

Two preparations of human IVlg were used in these experiments. Pentaglobin

(Biotest A.G., Dreieich, Germany), consisted of IgG (approximately 76%), IgM

(approximately 12%), and IgA (approximately 12%). Iveegam (Immuno U.S.,

Rochester, MI), contained IgA (0.2 fig/ml) and IgG (>95%). Both preparations were

depleted of anti-a Gal Ab by immunoadsorption using synthetic aGal columns

(Alberta Research Council, Alberta, Canada).

Flow Cytometric Analysis

Baboon blood samples were obtained, water lysed, and washed twice. For

determination of fluorescent latex bead clearance kinetics, the FACScan (Becton

Dickinson, Franklin Lakes, NJ) was calibrated to sample a fixed volume over a given

period of time (60fil ± 7fillminute). Freshly prepared fluorescent latex beads were

used as positive controL For the detection of pig cells, 1 0 ~l human immunoglobulin

was added to 1 x 106 cells to block non-specific binding of subsequently added

monoclonal Ab to Fe-receptors. The cells were then stained in 100 IJ-l F ACS medium

(1 % bovine serum albumin and 0.1 % azide in phosphate-buffered saline) using the

following conjugated monoclonal antibodies (mAb): W6/32 FITC (mouse anti

primate MHC Class I, IgG), I030HI-19 PE (pan-pig, mOuse anti-porcine leukocyte,

IgM), 36-7-5 FITC (mouse anti-mouse MHC Class I, IgG) and 12-2-2 PE (mouse

anti-mouse MHC class I, IgM), the latter two as isotypic controls. Dead cells were

41


excluded based on propidium iodide staining. Acquisition was performed under hi~

flow and samples were analyzed using WinList (Verity Software House, Topsham,

ME).

Polymerase chain reaction

A peR assay that amplifies the porcine cytochrome-B gene was used to detect pig

microchimerism in baboon samples. An internal PCR standard was created by adding

the porcine cytochrome-B primer sequence (Cyt-Fl) to either side of a fragment of

human A-2 phospholipase followed by cloning into a plasmid vector and quantitation

using limiting dilution PCR. Peripheral blood and BM cells were purified using

Histopaque 1077 (Sigma). The cells (2 - 10 x 105) were pelleted and frozen at -80°C

until use. Genomic DNA was extracted using the Qiamp Blood Kit® (Qiagen,

Valencia, CAl, quantified using Hoechst 33258 dye and a Hoeffer TKO-IOO

Fluorometer (Hoeffer Scientific Instruments, San Francisco, CA), and denatured. The

microchimerism assay consisted of 250 ng of sample DNA added to a total PCR

reaction volume of 100 JlL The final reagent mixture consisted of IX GeneArnp®

PCR Buffer II, 2.0 mM MgCIz, O.4mM dNTP, and 2.5 units of Amplitaq Gold®

(Perkin-Elmer, Philadelpllla, PAl. Eighty picomoles each of Cyt-Fl and Cyt-Rl

primers were added to the reactions. The PCR tubes were placed in a Perkin-Elmer

GeneAmp 9600 Thermal Cycler. The first denaturing step was 9 minutes at 95°C

followed by 40 cycles of 96°C for 10 seconds, 59°C for 30 seconds, and noc for 30

seconds. The program concluded with a 5 minute incubation at 72°C. The internal

standard was added to PCR reactions along with sample DNA to produce a 240 bp

product that was compared to the 210 bp product produced by the porcine

cytochrome-B gene through ethidium bromide-stained agarose gels. Samples that

produced a cytochrome-B product that was greater than or equal to the product from 5

copies of the standard were considered to be positive for microchimerism.

Hematology and Chemistry Parameters

Serial blood samples were taken for measurement of platelet count, prothrombin time,

partial thromboplastin time, fibrinogen, and fibrinogen degradation products

42


(Hematology Laboratory, Massachusetts General Hospital). Serum samples were

obtained for the measurement of lactate dehydrogenase (LDH) and basic renal and

liver function tests (Chemistry Laboratory, Massachusetts General Hospital).

Experimental Groups

All baboons were monitored for toxic side effects, and hematological parameters and

serum chemistries were measured.

Preliminary studies (n=4). The effect of ML on macrophages was assessed by

measuring the clearance kinetics of fluorescent latex beads from the circulation in

baboons. Fluorescent latex beads (lxI010) were infused iv on day 0 into baboons that

were either untreated, had received blank liposomes, or had received ML at various

dosages (200 mg twice daily x 2; 200 mg twice daily x 6; 800 mg once daily x I; or

800 mg twice daily x 2). Treatment with ML was timed such that the last dose was

administered on day -1 before bead infusion. In one experiment, when treatment was

continued for 6 days, ML was administered daily from days -2 to 3. Baboons received

25mg diphenhydramine prior to ML or blank liposomes administration to prevent

potential allergic reactions. Blood was drawn at various time points (0,1,5,15,30,

60, 120 and 240 minutes) after the beads were administered and flow cytometric

analysis was performed (as described earlier) to determine the number of fluorescent

latex beads remaining in the circulation.

Group I (controls, n~5). Baboons underwent the NMCR with transplantation of a

total of2-4 x 10 10 pPBPClkg on days 0 - 2. No ML or IVlg was administered.

Group 2 (n~2). Baboons underwent the NMCR with transplantation of a total of 2-4 x

1010 pPBPC/kg on days 0 - 2 or 0 - 3. Additionally, one baboon (BI30-94) received

ML at 40 mg/kg on days -2 and -I, followed by 20 mg/kg on days 1,3, and 4, and 40

mg/kg on day 7. A second baboon (B57-309) received ML at 80 mg/kg on days -2

and -I.

43


Group 3 (n~2). Baboons underwent the NMCR with transplantation ofa total of2-4 x

10 '0 pPBPClkg on days 0 - 2 or 0 - 3. Additionally, one baboon (B68-11) received

IVIg at 500 mg/kg/day from days 0 - 8, with a double dose (total 1000 mglkg) on day

I. A second baboon (B68-55) received IVIg at 500 mg/kg on days 0 - 2. IVIg was

administered immediately before pPBPC transplantation on each occasion.

RESULTS

Preliminary studies

Clearance of fluorescent latex beads from the circulation in an untreated baboon and

in a baboon that had received blank liposomes occurred within 4 hours (Fig. 1). In

contrast, treatment with ML led to substantially delayed clearance of fluorescent latex

beads. Four hours foIlowing treatment with one dose of ML at 800 mg, 5% of the

beads remained in the circulation, but after 24 hours, >99% of the beads had been

cleared. Administration of ML at 200 mg twice daily x 2 resulted in 6% persistence of

fluorescent latex beads 4 hours following infusion, and 5% after 24 hours. With this

regimen, the beads remained in the circulation for 4 days. When ML were infused at

200 mg twice daily x 6, 7% of the beads could be detected after 4 hours, with 4%

remaining after 24 hours, and 2% detectable after 5 days. \\!hen ML were

administered at 800 mg twice daily x 2, 28% of the fluorescent latex beads could be

detected after 4 hours, 17% after 72 hours, and <1 % at 12 days.

AIl baboons that received ML developed elevated LDH levels (maximum

approximately 3000 VII). The increase in LDH closely correlated with the dose and

duration of treatment of ML. This was probably related to release of LDH by dying

macrophages. No major changes were observed in renal, liver, or hematological

parameters.

These observations demonstrate that fluorescent latex beads are effectively removed

from the circulation in untreated baboons, and that treatment with ML results in

delayed clearance. In turn, this indicates a decrease of phagocytic function that is

most likely due to depletion of macrophages.

44

Depletion Qf macrophages

100000

Untreated

<-Blank liposomes ..... 200 mg bid x2 } 10000 200 mg bid x6 ML -0- 800 mg qd x1 - ,- 800 mg bid x2

TIME (minutes)

Figure 1 (Preliminary studies).

Clearance kinetics of fluorescent latex beads infused into baboons (data are presented in log scales).

The number of beads in circulation at I minute post infusion is designated as the baseline (100%). In

untreated baboons, or baboons that received blank liposomes, fluorescent latex beads were cleared

from circulation within 4 hours. In contrast, all baboons receiving medronate liposomes (ML)

demonstrated a substantially delayed clearance ofthe beads from the circulation. Treatment with ML at

200 mg or 800 mg twice daily x2 doses gave the most optimal results, with, respectively, 6% and 28%

persistence of fluorescent latex beads in the circulation 4 hours following infusion, 2% and 28%

remaining after 48 hours, and <1 % and 17% remaining after 72 hours. Treatment with 6 doses of ML

led to 7% persistence of the beads after 4 hours, 1% after 72 hours, and 3% after 72 hours. No beads

were detectable 5 - 12 days after administration in any baboon receiving ML.

45

Section Il- Chapter 2

Group 1 (controls)

These baboons received the NMCR with transplantation of a total of 2 - 4 X 1010

pPBPCikg on days 0 - 3 without ML or IVlg administration. The percentage of

porcine cell chimerism as detected by flow cytometry is depicted in Figure 2. On

average, pig cells made up approximately 14% (0.5% - 50%) of circulating cells 2

hours following transplantation of I x 1010 pPBPC I kg. Eighteen hours after pPBPC

transplantation, virtually no pig cells could be detected. This pattern was observed

after each day of pPBPC transplantation. After day 3, no porcine cells could be

detected.

100

75

50

25

Figure 2.

~857-323

857-56

868-54

~857-301

-0- 869-393

Pig cell chimerism detected by flow cytometry in 5 baboons in Group 2, using a pan-pig leukocyte

marker. All baboons received the NMR with transplantation of approximately 2 - 4 xlO lo pPBPClkg

divided equally over days 0, 1, and 2. No medronate liposomes or IVIg was administered. Horizontal

bars indicate the periods during which pPBPC were infused. Arrows indicate sampling timepoints 2

hours after transplantation. Two hours after transplantation of pPBPC on day 0, a mean of 14%

circulating pig cells was detected. Eighteen hours later (marked by timepoint 1), virtually no pig cells

(1.1%) were detectable. On day 1, 2 hours following transplantation, pig cell chimerism reached

iO.7%, falling to 0.9% eighteen hours later. On day 2, 17.4% of all cells were of porcine origin 2 hours

post transplantation, decreasing to near-undetectable levels (1.2%) on day 3. By day 4, no pig cens

were detectable.

46


Porcine DNA was detected by PCR continuously in the blood for approximately 19 (7

- 28) days and intermittently up to 263 days. Porcine DNA was detected

intermittently in the bone marrow in 4 out of 5 baboons between 16 and 365 days.

All baboons developed a thrombotic microangiopathic state, with elevated LDH

(>1,500 U/I) and thrombocytopenia «20,000 I~l) necessitating platelet transfusions

in most instances. This state of microangiopathic thrombosis has been described and

discussed elsewhere (30).

These data indicate that, using this NMCR, transplanted pPBPC are rapidly cleared

from the circulation of baboons.

Group 2

In addition to the NMCR, these two baboons received ML before, or during, pPBPC

transplantation. In B 130-94, 2 hours after the first pPBPC transplantation, pig cells

made up 49% of the cells detected in the circulation (Fig. 3). The following morning,

this percentage had increased to 53%. The percentage of pig cells increased to reach a

maximum of 78% 2 hours after the second pPBPC transplantation (Fig. 4), after

which pig cell chimerism was slowly lost, despite continued administration of ML. A

last peak of circulating pig cells was observed on day 4, after the final IxlOlo

pPBPClkg were transplanted. After day 5, no pig cells were detectable. This baboon

died of infectious complications on day 12. A similar pattern was observed in B57-

309, where 2 hours after the first transplantation of pPBPC, 30% of circulating cells

were of porcine origin. The following day, this percentage was sustained at 30%. Two

hours after the second transplantation, the percentage of pig cells increased to 33%

and reached a maximum of 55% 18 hours later. After the third, and last, day of

pPBPC transplantation, however, the percentage of pig cells decreased to 25% and, by

day 4, no pig cells were detected.

Porcine DNA was detected continuously for 16 days in the blood of B57-309 and

intermittently through day 96. No porcine DNA was detected in the bone marrow of

47

Section II - Chapter 2

this baboon. Porcine DNA was detected in all tissues of B130-94 at autopsy on day

12.

Both baboons developed increased LDH levels (7900 VII and >10,000 U/l for B130-

94 and B57-309, respectively), probably limiting any potential increase in the dosage

of ML. Furthennore, a microangiopathic state similar to that in Group 1 was

observed, with a marked thrombocytopenia «20,000 I~l), necessitating platelet

transfusions.

Treatment with ML, therefore, led to substantially higher initial levels of circulating

pig cells, and sustained these cells in the circulation for 4 - 5 days.

Group 3

These two baboons underwent the NMCR before pPBPC transplantation, and also

received daily infusions of IVlg. In B68-11 the initial percentage of circulating

porcine cells (9.3% at 2 hours after the first transplantation ofpPBPC) was equivalent

to that in the Group 2 baboons (Fig. 5). Two hours after the second transplantation of

pPBPC (day 1),4.4% of the cells were of porcine origin. Eighteen hours later, this

percentage had increased to 7.4%. There was, therefore, slightly better maintenance of

pig cells in this baboon than in the Group 2 baboons. Over the next days, however,

pig cells were lost from the circulation. In the second baboon (B68-55), the

percentage of pig cells was also similar to that of the Group 2 baboons during the first

two days of pPBPC transplantation (2 hours post transplantation, 8% and 12%,

respectively). Before the third day of pPBPC transplantation, 5% porcine cells

remained detectable. After the third transplantation of pPBPC, this baboon developed

a leukocytosis of 90,000 I~l, of which 70% (63,000 I~l) were of porcine origin. The

baboon died suddenly 2 hours after the third transplantation of pPBPC. Autopsy

revealed a widespread thrombotic microangiopathy, with thromboses in the

microvasculature of the heart, lungs, and kidneys. These findings were consistent with

previous findings when death occurred after high-dose pPBPC transplantation (30).

48

100

<f? 75

.21 E 0. .!!l i ~ 50 0..5

.s:: U - 25

Figure 3.

3


... - B130-94

~B57-309

Da s

5

Pig cell chimerism detected by flow cytometry in 2 baboons in Group 3, using a pan-pig leukocyte

marker.

B130-94 received the standard NMR with transplantation of approximately 2 - 4 xlOlO pPBPClkg

divided over days 0, I, 2, and 3. Horizontal bars indicate periods of pPBPC transplantation. Arrows

indicate sampling timepoints 2 hours after transplantation. B130-94 received medronate liposomes

(ML) at 40 mgikg on days -2 and -I, followed by 20 mg/kg on days I, 3, and 4, and 40 mg/kg on day

7. Two hours after pPBPC transplantation on day 0, pig cells made up 49% of the total cells in the

circulation. Eighteen hours later, this percentage had increased to 53%. Two hours after transplantation

ofpPBPC on day 1, the pig cell percentage increased to 78% (see Fig. 4). On days 2 and 3, even after

transplantation of more pPBPC, pig cell chimerism was lost. On day 4, after a further Ix1 010 pPBPCikg

were transplanted, a final peak of pig cells was observed. These cells were rapidly cleared from the

circulation and, after day 5, no pig cells were detectable.

B57-309 underwent the NMR and pPBPC transplantation on days 0, I, and 2 (to a total of 3 xl0 10

pPBPC/kg) and received ML at 80 mg/kg on days -2 and -\. Two hours after pPBPC transplantation

on day 0, 30% of cells were of porcine origin. The following day, this percentage was sustained at 30%

and increased to 33% two hours following pPBPC transplantation on day 1 and to 55% eighteen hours

later. After pPBPC transplantation on day 2, the percentage of pig cells decreased to 25% and, by day

4, no pig cells remained.

49

Section 1/ - Chapter 2

'0 .. -= C <.I

~ <=

OS .. '" z

1

Figure 4.

0.03%

;

;

;

.

A

.,

B

.. ro,"'----------------T-----

.' .

'Q. = OS

l>.

78%

Baboon MHC Class I

·· .... ,.t

Two-color flow cytomerric analysis of circulating cells in B 130-94 , 2 hours after pPBPC

transplantation on day 1. Staining was performed for baboon MHC Class I (W6/32 FITC), negative

isolype control (1 2.2.2 PE, Fig. A), and pan~pig leukocyte (1030HI 19 PE, Fig. B). At this timepoint,

78% of cells were of porcine origin.

Porcine DNA was detected in the blood of 868·11 continuously through day 9 and

intermittently through day 43. [n the bone marrow, porcine DNA was detected only

on day 14. All tissues ofB68·55 at autopsy on day 2 contained porcine DNA.

868·11 developed a thrombotic microangiopathic state, with elevated LDH (> 1,500

U/I) and thrombocytopenia (<20,000 IIlI ) comparable to Group I baboons.

The addition of IVlg to the NMCR, therefore, led to a marginally delayed rate of

clearance of pPBPC and. in one case, to a very high level of chimerism (that was

probably a major factor in the death of the baboon). These differences may be

explained by differences in the preparations of IVIg used. Alternatively, treatment

with IVlg in 868·55 may have been more effective in saturating all Fe·gamma

receptors of the macrophages, thereby preventing opsonization and phagocytosis of

porcine ce ll s, and leading to higher chimerism.

50

Depletion o/macrophages

100

~

?!< 75

.52' E 1:1. .!Il l; ~ 50 1:1..5

..c:: o ~ 25

t B68-11

~B68-55

........ ~... Days O~--~~----,-~--,-----,-----,

o SlliB't t 2 ±R1iBt

3 4 5

Figure 5.

Pig cel! chimerism detected by flow cytometry in 2 baboons in Group 4, using a pan*pig leukocyte

marker. Both baboons received the NMR with transplantation of approximately 2 - 4 xl 010 pPBPClkg

divided over days 0, 1, and 2. Horizontal bars indicate period of pPBPC transplantation, arrows

indicate sampling timepoints 2 hours after transplantation. B68-11 received IVlg at 500 mglkglday

from days 0 - 8, with a double dose (total 1000 mgikg) on day 1. The initial pig cell chimerism 2 hours

post pPBPC transplantation on day 0 was 9.3%, which decreased J 8 hours later to 1.6%. Two hours

after pPBPC transplantation on day 1, 4.4% of cells were of porcine origin, with an increase to 7.4%

eighteen hours later. Pig cell chimerism decreased and became undetectable over the next few days.

B68-55 received IgM-containing IVlg (Pentaglobin) at 500 mglkg on days 0 - 2. After the first 2 days

ofpPBPC transplantation, pig cells were detected at 8% and 12%, respectively. On day 2, before the

last transplantation ofpPBPC, pig cell chimerism was sustained at 5%. Two hours after transplantation

of pPBPC on this day, the pig cell chimerism was 70%, with a leukocytosis of 90,000 /J.d, at which

time the baboon died from thrombotic complications.

DISCUSSION

The protocols developed at our laboratory aimed at inducing immunological

tolerance through establishing stable mixed hematopoietic chimerism have not proven

successful in the discordant pig-to-baboon model (31). Our NMCR, which includes

non-myeloablative irradiation, T cell depletion, costimulatory blockade, complement

51

Section I1- Chapter 2

depletion, and (temporary) depletion of xenoreactive anti-aGal Ab, has thus far been

insufficient to allow engraftment of pPBPC in baboons.

It has been suggested that macrophages may pose barriers to hematopoietic

engraftment of xenogeneic cells, and that depletion of macrophages can improve

chimerism in human-to-SCID-mouse xenotransplantation models (14,32). We are the

first to report successful depletion of macrophages without significant side-effects in a

large animal model using ML. Furthermore, to assess the negative effect of the MPS

on the induction of chimerism in the pig-to-baboon model, we tested macrophage

depletion with ML, or inhibition of the ability of macrophages to opsonize and

phagocytose particles with IVlg, in our NMCR.

When ML were added to our protocol, and to a lesser extent when IVlg was added,

substantially higher initial levels of circulating pig cells were observed, and pig cells

were sustained in the circulation for longer periods of time. These data indicate that

macrophages, indeed, play an important role in the clearance of porcine cells from the

circulation of baboons. This short-term improvement in pig cell survival, however,

did not lead to engraftment of these cells or to the development of immunological

tolerance. Experiments in rodents indicate that depletion of macrophages with ML is

temporary, and that a rebound in the number and/or the activity of macrophages can

occur within days of discontinuing ML therapy (Cheng J, et aI., manuscript in

preparation). It is not clear, therefore, whether depletion of macrophages for only a

limited period of time will be sufficient to establish hematopoietic cell chimerism.

One may postulate that macrophages will need to be depleted for an extended period

of time, sufficient to allow the porcine cells to home to and engraft in the recipient

baboon's bone marrow.

One of the mechanisms through which baboon macrophages may clear porcine cells is

through antibody-dependant cellular cytotoxicity. Although anti-aGal Ab can be

efficiently removed from the circulation (33), continued production of anti-aGal Ab

cannot be prevented (34). This is illustrated by the observation that, within hours of

extracorporeal immunoadsorption of antiaGal Ab and pPBPC transplantation, the

52


pPBPC in the circulation are largely coated with anti-aGal IgM and IgG Ab (Awwad

M, et a!., unpublished data). Coating of pPBPC by anti-aGal IgG may facilitate

phagocytosis ofpPBPC by cells of the MPS (i.e., by macrophages), thus reducing the

potential of engraftment. Methods that successfully maintain depletion of anti-a Gal

Ab, perhaps only for a limited period of time, may, therefore, be of benefit. However,

it is possible that pPBPC may be phagocytosed by macrophages in the absence of

coating.

Depletion of cells of the MPS may, however, be counterproductive, as these cells may

be required to transport porcine antigen to the thymus if central tolerance is to be

induced (35). Furthennore, recent data from our laboratory suggest that macrophages

are essential in maintaining the state of humoral unresponsiveness to porcine antigens

induced by treatment with anti-CDl54 mAb (BubIer L, et a!., manuscript in

preparation), and may thereby be associated with the induction of peripheral

tolerance. Depletion of macrophages resulted in an elicited Ab response to porcine

antigens following hematopoietic cell transplantation that was previously prevented

with costimulatory blockade with anti-CD 154 mAb. Sustained depletion of anti-aGal

Ab, macrophages, and T cells may allow hematopoietic chimerism and

immunological tolerance to be achieved.

In addition to the effect IVIg may have on macrophages, other mechanisms of action

of IVIg may be beneficial in pig-to-primate xenotransplantation. Proposed actions of

IVIg include reducing complement-mediated injury by modifying the antibody

antigen ratio (23) and, particularly for IgM-containing IVIg (such as Pentaglobin), by

'scavenging' of activated complement fragments (36). Furthermore, down-regulation

of B cells by binding to B cell immunoglobulin, functioning as anti-idiotypic

antibodies and binding ofxenoreactive antibodies (37,38), or binding to IgG receptors

on CDS+ T cells, thereby altering T-helper functions (39), have also been detennined.

In conclusion, it appears that cells of the MPS play an integral role in the clearance of

porcine cells from the circulation of baboons. Depletion of the cells of the MPS, or

inhibition of their function, leads to higher levels of chimerism that are better

53

Section Il- Chapter 2

sustained, and may potentially facilitate the induction of mixed hematopoietic

chimerism and immunological tolerance to porcine antigens in baboons.

REFERENCES

1 Sharabi Y and Sachs DH. Mixed chimerism and permanent specific transplantation tolerance induced by a non~!ethal preparative regimen. J Exp Med 1989; 169: 493

2 Lee LA, Sergio JJ, Sykes M. Natura! killer ce!!s weakly resist engraftment of allogeneic long~term mUltilineage-repopulating hematopoietic stem cells. Transplantation 1996; 61: 125

3 Wekerle T, Sayegh MH, Hi!! J, et al. Anti-CD 154 or CTLA4Ig obviates the need for thymic irradiation in a non-myeloablative conditioning regimen for the induction of mixed hematopoietic

chimerism and tolerance. Transplantation 1999; 68: 1348 4 Wekerle T, Kurtz J, Ito H, et al. Allogeneic bone marrow transplantation with co-stimulatory

blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat Med 2000;6:464

5 Huang CA, Fuchimoto Y, Scheier-Dolberg R, et al. Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model. J Clin Invest 2000; 105:173

6 Kawai T, Cosimi AB, Colvin RB, et al. Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 1995; 59: 256

7 Kimikawa M, Sachs DH, Colvin RB, et al. Modifications ofthe conditioning regimen for achieving mixed chimerism and donor-specific tolerance in cynomolgus monkeys. Transplantation 1997; 64:

709 8 Sharabi Y, Aksentijevich I, Sundt III TM, et al. Specific tolerance induction across a xenogeneic

barrier: production of mixed rat/mouse lymphohematopoietic chimeras using a nonlethal preparative regimen. J Exp Med 1990; 172: 195

9 Ildstad ST, Wren SM. Cross-species bone marrow transplantation: evidence for tolerance induction, stem cell engraftment, and maturation ofT lymphocytes in a xenogeneic stromal environment (rat-> mouse). J Exp Med 1991; 174: 67

10 Ko DS, Bartholomew A, Poncelet AJet al. Demonstration of multiline age chimerism in a nonhuman primate concordant xenograft model. Xenotransplantation 1998; 5: 298

II Alwayn IP, van Bockel HJ, Daha MR, et al. Hyperacute rejection in the guinea pig-to-rat model without formation of the membrane attack complex. Transpllmmunol 1999; 7: J 77

J2 Qian Z, Wasowska BA, Behrens E, et a1. C6 produced by macrophages contributes to cardiac allograft rejection. Am J Pathol 1999; 155: 1293

13 Leonard CT, Soccal PM, Singer L, et al. Dendritic cells and macrophages in lung allografts. A role in chronic rejection? Am J Respir Crit Care Med 2000; 161: 1349

14Terpstra W, Leenen P J, van den Bos C, et al. Facilitated engraftment of human hematopoietic cells in severe combined immunodeficient mice following a single injection of Cl2MDP liposomes. Leukemia 1997; II: J049

15 Fox A, Koulmanda M, Mandel TE, et al. Evidence that macrophages are required for T-cell infiltration and rejection offetal pig pancreas xenografts in nonobese diabetic mice. Transplantation 1998; 66: 1407

16 Wu G, Korsgren 0, van Rooijen N, et al. The effect of macrophage depletion on delayed xenograft rejection: studies in the guinea pig-to-C6-deficient rat heart transplantation model. Xenotransplantation. 1999 Nov;6(4):262

17 Feng X, Zheng XX, Yi S, et al. IL-10/Fc inhibits macrophage function and prolongs pancreatic islet xenograft survival. Transplantation 1999; 68: 1775

18 van Rooijen N, van Nieuwmegen R. Elimination of phagocytic cells in the spleen after intravenous injection of liposome-encapsulated dichloromethylene diphosphonate. An enzyme~histochemical study. Cell Tissue Res 1984; 238: 355

19 Van Rooijen N, Sanders A. Liposome mediated depletion of macro phages: mechanism of action, preparation of liposomes and applications. J Immunol Methods 1994; 174: 83

20 Van Rooijen N. Minireview: The liposome-mediated macrophage suicide technique. J Immunol Methods J 989: 124; 1

54


21 Fehr J, Hofmann Y, Kappeler U. Transient reversal of thrombocytopenia in idiopathic thrombocytopenic purpura by high-dose intravenous gamma globulin. N Eng! J Med 1982; 306: 1254

22 Bussel JB. Treatment effects on chronic idiopathic thrombocytopenic purpura. In: Imbach P; ed. Immunotherapy with Intravenous Immunoglobulins. San Diego: Academic Press 1991; 253

23 Dalakkas MC, Illa I, Dambrosia JM, et a1. A controlled trial of high-dose intravenous immune globulin infusions as treatment for dermatomyositis. N Engl J Med 1993; 329: 1993

24 Jordan SC, Quartel A W, Czer LS, et al. Posttransplant therapy using high-dose human immunoglobulin (intravenous gammaglobulin) to control acute humoral rejection in renal and cardiac allograft recipients and potential mechanism of action. Transplantation 1998; 66: 800

25 Furth S, Neu AM, Hart J, et al. Plasmapheresis, intravenous cytomegalovirus-specific immunoglobulin and reversal of antibody-mediated rejection in a pediatric renal transplant recipient: a case report. Pediatr Transplant 1999; 3: 146

26 Singer JM, Adlersberg L, Hoenig EM, et al. Radiolabeled latex particles in the investigation of phagocytosis in vjvo: clearance curves and histological observations. J Reticuloendothel Soc 1969; 6:561

27 Kozlowski T, Shimuzu A, Lambrigts 0, et al. Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption. Transplantation 1999; 67: 18

28 Alwayn IPJ, Appel JZ III, Goepfert C, et a1. Inhibition of platelet aggregation in baboons: therapeutic implications for xenotransplantation. Xenotransplantation 2000; 7: 247.

29 Nash K, Chang Q, Watts A, et al. Peripheral blood progenitor cell mobilization and leukapheresis in pigs. Lab Anim Sci 1999; 49: 645

30 Buhler L, Basker M, Alwayn IPJ, et al. Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Transplantation 2000; 70: 1323.

31 Kozlowski T, Monroy R, Giovino M, et al. Effect of pig-specific cytokines on mobilization of hematopoietic progenitor cells in pigs and on pig bone marrow engraftment in baboons. Xenotransplantation 1999; 6: 17

32 Yerstegen MM, van Hennik PB, Terpstra W, et al. Transplantation of human umbilical cord blood cells in macrophage-depleted scm mice: evidence for accessory cell involvement in expansion of immature CD34+CD38· cells. Blood 1998; 91: 1966

33 Watts A, Foley A, Awwad M, et al. Plasma perfusion by apheresis through a Gal immunoaf1mity colwnn successfully depletes anti-Gal antibody - experience with 320 aphereses in baboons. Xenotransplantation 2000; 7: 181.

34 Alwayn IP, Basker M, Buhler L, et a!. The problem of anti-pig antibodies in pig-to-primate xenografting: current and novel methods of depletion and/or suppression of production of anti-pig antibodies. Xenotransplantation 1999; 6: 157

35 Wekerle T and Sykes M. Mixed Chimerism as an approach for the induction of transplantation tolerance. Transplantation 1999; 68: 459

36 Rieben R, Roos A, Muizert Y, et al. Immunoglobulin M-enriched human intravenous immunoglobulin prevents complement activation in vitro and in vivo in a rat model of acute inflammation. Blood 1999: 93: 942

37 Dietrich G and Kazatchkine MD. Normal immunoglobulin G (lgG) for therapeutic use (IntTavenous Ig) contain antiidiotypic specificities against an immunodominant, disease-associated, cross-reactive idiotype of human anti-thyroglobulin autoantibodies. J Clin Invest 1990; 85: 620

38 Nydegger UE, Sultan Y, Kazatchkine MD. The concept of anti-idiotypic regulation of selected autoimmune diseases by intravenous immunoglogulin. Clin Immunol Immunopathol1989; 53:S72

39 Ruiz de Souza Y, Carreno MP, Cavailon JM, et al. Modulation of cytokine production by mmunoglobulins for intravenous use (IYlg). F ASEB J 1984; 8: 1238

55


56

III The importance of snppressing xenoreactive antibody production

3 Curreut and novel methods of depletion andlor suppression of antipig antibodies

Adaptedfrom: Alwayn IPJ, Basker M, Buhler L, and Cooper DKC. The problem of anti-pig antibodies in pig-to-primate xenografting: current and novel methods of depletion and/or suppression of production of anti-pig antibodies. Xenotransplantation 1999;6: 157-68


ABSTRACT

The role of antibodies directed against Galal-3Gal (aGal) epitopes in porcine-to

primate xenotransplantation has been widely studied during the past few years. These

antibodies (anti-aGal) have been associated with both hyperacute rejection and acute

vascular rejection of vascularized organs. Depletion and (temporary or pennanent)

suppression of production of anti-aGal seems essential for obtaining long-tenn

survival of these organs, even when the ultimate aim is the achievement of

accommodation or tolerance. Although greater than 95% depletion of anti-aGal can

be realized by the use of immunoaffinity column technology, to date no regimen has

been successful in preventing the return of anti-aGal from continuing production. In

this review, we discuss current and novel methods for achieving depletion or

inhibition (i.e., extracorporeal immunoadsorption, anti-idiotypic antibodies, the

intravenous infusion of immunoglobulin or oligosaccharides) and suppression of

production (i.e., irradiation, pharmacologic agents, specific monoclonal antibodies,

immunotoxins) of anti-aGal antibodies.

INTRODUCTION

In 1966, Perper and Najarian were among the first to recognize that both antibodies

and complement were involved in the hyperacute rejection (HAR) of organs

transplanted berureen widely disparate species, such as pig-to-dog (1). It has been

knO\vn for more than 10 years that pigs and other nonprimate mammals express

tenninal Galal-3Gal oligosaccharides (aGal) as surface antigens on many cells,

including the vascular endothelium (2). Galili et a1. showed in 1984 that humans

possess antibodies that have anti-a Gal specificity (3), and that its IgG component

comprises 1 % of circulating IgG in human serum. It was not until 1991, however, that

Good et al. demonstrated that these natural antibodies (anti-aGal) playa major role in

the HAR of pig xenografts in primates (4-6). Furthermore, they determined that anti

aGal accounts for more than 85% of circulating natural anti-pig antibody in humans

and that these antibodies are primarily of IgG and IgM subclasses (4-8). Galili and

60

Depletion of anti-pig antibodies

coworkers have postulated that anti-aGal develop soon after birth, similar to ABO

antibodies in humans, due to exposure to colonizing microorganisms in the

gastrointestinal tract that express aGal surface antigens (9). There is now clear

evidence that anti-aGal are responsible for complement-mediated HAR of porcine

vascularized organs transplanted into human or nonhuman primate recipients (4-

6,10,11).

Even if complement activation is inhibited (e.g. by the use of complement regulatory

agents or porcine organs transgenic for human complement regulatory proteins), anti

aGal still plays a major role in a delayed rejection response, variously termed delayed

xenograft rejection or acute vascular rejection (AVR), which is most probably

complement-independent (12,13). Since it is now widely accepted that pigs (that

express Gal epitopes in abundance) are the most likely animals to provide organs for

xenotransplantation in humans (who have high levels of anti-aGal), the importance of

the aGal antigen-anti-aGal antibody complex in xenotransplantation cannot be

overemphasized.

In this chapter, current knowledge of anti-aGal and its importance in the rejection of

vascularized organs in the pig-to-primate model is discussed. We summarize various

methods being explored of achieving depletion or inhibition of anti-aGal and,

furthermore, of suppressing production.

Anti-aGal Antibodies

Oriol et al. have presented detailed data on the presence of aGal epitopes in different

porcine tissues (14) and have demonstrated that aGal is expressed unifonnly on

vascular endothelium of all pig breeds (15). This contrasts with humans, who express

ABO blood group epitopes rather than aGal, even though both humans and pigs

express N-acetyllactosamine and a sialic acid on their vascular endothelium (Chapter

1, Table 1) (16). It has long been considered that anti-aGal and anti-A and anti-B

blood group antibodies are members of the same family of natural antibodies (17),

and further evidence to this effect has recently been presented by Parker et al (18).

61

Section 1II- Chapter 3

The concentration of aGal on most pig cells approximates 107 epitopes per cell (2).

The IgM subclass of anti-aGal is of special importance in BAR since IgM is often

found to be deposited on graft endothelium before, and sometimes in the absence of,

IgG (10,19), In addition, depletion of anti-aGal IgM eliminates complement-mediated

cytotoxicity of human serum on porcine endothelial cells (20). However, evidence has

also been presented that IgG can playa role in HAR as well as in A VR (11,21),

Humans and nonhuman primates (with the exception of New World monkeys) have a

nonfunctional a-galactosyltransferase pseudogene, hence lack the encoded al,3-

galactosyltransferase enzyme, and therefore do not make aGal (22). In the absence of

aGal, primates develop anti-aGal. It has been suggested that nonhuman primates

were exposed to an aGal-expressing pathogen at an evolutionary stage after the

divergence of New from Old World monkeys, and that the survival of Old World

primates depended on adapting by suppressing aGal expression and producing anti

aGaI antibody (23), Since aGaI epitopes have been fonnd on the surface of certain

parasites, bacteria, and viruses (reviewed in 6), anti-aGal may playa role in defense

mechanisms against these organisms in humans and nonhuman primates. These

observations suggest that anti-aGal may be essential to primates in providing natural

immunity to a wide variety of pathogens.

Anti-aGal Antibodies: Role in Hyperacute Rejection

'When vascularized organs are transplanted between pigs and primates, HAR almost

nniformly occurs (24), The development ofHAR in the pig-to-primate model depends

on the activation of complement, mostly, if not entirely, through the classical

pathway, Activation of this pathway is initiated by the binding of anti-aGal to the

corresponding antigens (Le, aGal) (19,25). When anti-aGal is depleted from

xenograft recipients, prolongation of graft survival is seen (26-29). \\'hen porcine

hearts are transplanted into newborn baboons (that lack anti-aGal), HAR does not

occur (30). The importance of the aGal antigen-anti-aGal antibody system was

confirmed by Collins et ai, who demonstrated that HAR occurred when an organ from

62


a New World monkey (expressing aGal) was transplanted into a baboon (that

produces anti-aGal) (31).

Anti-aGal Antibodies: Role in Acute Vascular Rejection

HAR can be prevented by antibody depletion or inhibition (26-29), complement

depletion or inbibition (32,33), or by transplanting organs from pigs transgenic for

human complement regulatory proteins (34-37). However, a delayed rejection

phenomenon (AVR) occurs and leads to destruction of the xenograft (13). It has been

proposed that this destruction results from the development of high levels of high

affinity antibody, largely IgG, directed against aGal and possibly also new pig

epitopes (12). This response occurs despite high doses of immunosuppressive therapy.

Increases in anti-aGal IgG of 100-300-fold have been documented after experimental

organ and bone marrow transplantation (38,39) and after clinical pancreatic islet cell

transplants (40). Even after the transplantation of relatively avascular porcine

meniscus cartilage to cynomolgus monkeys, anti-a Gal titers (IgM and IgG) rose 10-

fold (41). These induced antibodies appear to be the major cause of A VR (42,43).

Accommodation

It has been described that ABO- or human leukocyte antigen (HLA)-incompatible

allografts are able to survive despite the presence of circulating antibodies directed

against these determinants (44-46). This process has been termed accommodation,

and can be applied to any system in which antibodies exist that are potentially reactive

with antigens on the endothelium of a primarily vascularized organ graft and yet

where antibody-mediated rejection does not occur (47,48). The phenomenon has not

yet been documented to occur in Gal-incompatible large animal models. Several

possible explanations for the phenomenon of accommodation have been proposed

(47). Firstly. the returning antibodies may be different in isotype, affinity, and/or

specificity, and are thus unable to initiate rejection. Secondly, the surface antigens on

the vascular endothelium may show subtle changes during the absence of natural

antibodies. Finally, the endothelial cells may adapt during the return of antibodies and

respond differently or become 'desensitized'. Bach et al have presented evidence that

63

Section III - Chapter 3

changes occur in the endothelial cells, with upregulation of -·beneficial' genes and

downregulation of 'detrimental' genes (48).

Tolerance

Perhaps the ultimate method for avoiding HAR and A VR would be to induce B cell

tolerance. 1fT cell tolerance were also induced, the subsequent cellular response (and

the complications associated with long-term high-dose immunosuppression) would

also be avoided. This option is being studied at our center, and involves the induction

of mixed hematopoietic cell chimerism between pig and primate (38,39,49).

Tolerance is achieved when there is permanent specific unresponsiveness to donor

antigens (but not to other antigens) in the absence of maintenance immunosuppressive

therapy. To induce this state in the pig-to-primate model, a combination of (at least

temporary) (i) anti-aGal depletion or inbibition, (ii) depletion or inhibition of

complement, (iii) T cell depletion (and/or costimulatory blockade), and (iv) pig

hematopoietic cell engrafiment, would appear to be necessary. The use of organs from

genetically engineered pigs transgenic for human complement regulatory proteins

may also prove an advantage, although this remains uncertain.

'Whether accommodation is to be achieved or tolerance is to be induced, however, it

seems essential to devise strategies that lead to the depletion of anti-aGal and to the

suppression of production of anti-aGal for a period of time.

METHODS OF DEPLETION OR INHIBITION OF ANTI-aGAL

ANTIBODIES

Initial experiments III the 60s and 70s directed against removing xenoreactive

antibodies were performed by perfusion of the recipient's blood or plasma through a

donor-specific organ, such as a pig kidney or liver (50-53). Antibodies were adsorbed

by the perfused organ's vascular endothelium, thus temporarily depleting the anti

species antibody and enabling modestly prolonged survival of a subsequently

transplanted organ. Initial experiments in the pig-to-non-human primate model

(26,54) showed the pig liver to be a better immunoadsorbent than the kidney, a

64


finding confirmed recently by Azimzadeh et al (55). Evidence has also been presented

that perfusion of human blood through pig lungs is more effective in removing anti

pig antibody tlmn perfusion through pig liver or spleen (56). Other methods for

depleting anti-aGal include (i) plasma exchange, (ii) plasma perfusion through non

specific immunoadsorbants (e.g., protein columns), and (iii) plasma perfusion

through specific antibody sorbents (e.g., synthetic aGal immunoaffinity columns).

Plasma Exchange

Plasma exchange - the (complete) removal of the subject's plasma (with replacement

of volume by other fluids) - is utilized as a treatment for numerous conditions, such as

myasthenia gravis, thrombotic microangiopathies, and cryoglobulinemias. All or most

circulating antibodies, including anti-a Gal, are removed. Because of this temporary

state of agammaglobulinemia, the patient is at risk for infection. Furthermore, the

patient may be depleted of plasma proteins important to the coagulation cascade.

Alexandre and coworkers were successful in using repeated plasma exchange to

remove circulating anti-A and anti-B blood group antibodies from potential kidney

transplant recipients (45), and demonstrated that the phenomenon of accommodation

could result. This technique was also moderately successful in depleting xenoreactive

natural antibodies from baboons in a pig-to-baboon kidney transplant model (27) but,

although the porcine grafts functioned for up to 22 days, accommodation did not

occur, and the grafts were lost to A YR.

Nonspecific Antibody Sorbents

Nonspecific antibody adsorption differs from plasma exchange in that the plasma is

passed through an immunoaffinity column containing proteins that remove

immunoglobulins from the plasma. The remaining plasma is then returned to the

patient, reducing the need for replacement fluids. The proteins (such as

staphylococcal protein A or protein G) are efficient in removing antibody by binding

to the Fc portion of the antibody. Since the proteins are non-specific, all antibodies

are removed, resulting again in hypogammaglobulinemia and predisposition for

infectious complications. Palmer et al. reported some success in the use of protein A

65


columns to deplete anti-HLA antibodies in potential renal transplant recipients (46),

and this technique has been used by several groups in xenotransplantation studies.

Specific Antibody Sorbents

Specific antibody sorbents for anti-a Gal utilize columns that remove anti-aGal from

plasma by binding them to natural or synthetic aGal oligosaccharides. Significant

hypogammaglobulinemia does not occur. Initial studies were by Ye et al. (57), who

demonstrated that extracorporeal immunoadsorption (EIA, Figure 1) of baboon

plasma, utilizing immunoaffinity columns of melibiose on 4 consecutive days, could

reduce cytotoxicity to pig kidney (PKI5) cells to 20%.

Anti-alpha-Gal

Saline 1m m uno-coagulan

affinity fr colum n

Centrifuge

Jp,,,m, Access <- Cellular

elements

Return

Figure I.

Diagram of extracorporeal immunoadsorption circuit, with access through the internal jugular vein and

return through the saphenous vein.

Vlhen more specific aGal oligosaccharides became available, a course of EIA

resulted in successful depletion of anti-aGal for 4-5 days after which anti-aGal

returned and led to rejection of a transplanted porcine organ (28,29,58,59). Kozlowski

et al. (60) provided evidence indicating that EIAs should be carried out approximately

24 - 48 hours apart in order to enable equilibration of anti-aGal between the intra-

66

Depletion of anli-pig anlibodies

and extravascular spaces to occur. Although (lGal immunoaffinity columns are

efficient in depleting anti-aGal, in order to maintain depletion, other therapeutic

modalities need to be employed, such as inhibiting the production of anti-aGar. This

has proved much more difficult, and is perhaps the major barrier to successful

xenotransplantation facing us at the present time (see below).

Antiidiotypic Antibodies

An alternative approach to using a synthetic oligosaccharide column is the use of

antiidiotypic antibodies (AlA) directed against idiotypes expressed on anti~aGal

antibodies. AlAs recognize specific idiotypes that are antigenic determinants within

the variable regions of immunoglobulins. AlAs are also directed against the B

lymphocyte subpopulations that bear the same idiotypes as surface receptors. Koren et

al. (61) produced AlAs in mice by the injection into the mouse of human anti~pig

antibodies (eluted from pig organs after repeated petfusion with human plasma) and

by the subsequent creation of hybridomas. Several of these AlAs, when incubated

with human serum, had a major inhibitory effect on serum cytotoxicity towards pig

PK15 cells in vitro. The AlAs could be made up as an immunoaffinity column or

could be infused intravenously into the recipient (to be bound by anti~aGal, thus

inhibiting binding of anti~aGal to a transplanted organ). \\Then infused into baboons,

serum cytotoxicity was markedly reduced (to approximately 10%). Recently. we have

produced a pig polyclonal AlA by immunizing a pig with human anti-aGal

antibodies. The purified final preparation contained 1~2 % AlA. After repeated

administration to a baboon (following repeated EIA of anti-aGa!), a delayed return of

anti-a Gal and reduced cytotoxicity to pig cells was observed. Furthermore, at this

time point, the baboon serum was able to partially inhibit the cytotoxicity of other

highly cytotoxic sera.

Intravenous Infusion of Immunoglobulin

The intravenous (i. v.) infusion of concentrated human immunoglobulin (IVIG) has

been used successfully in the treatment of certain autoimmune disorders, e.g.,

idiopathic thrombocytopenic purpura (62), and more recently as therapy to reduce

67

Section JlI- Chapter 3

anti-HLA antibodies in highly-sensitized patients awaiting organ transplantation (63).

Magee et al. (20) demonstrated that the Lv. infusion ofinununoglobulin could prevent

the HAR of pig organs transplanted into baboons. Different hypotheses have been

proposed to explain the mechanism of action of IVIG, including the presence of AlAs

or complement inhibition. Recent studies suggest that IVIG accelerates the

physiological catabolism of IgG by saturating specific intracellular receptors,

allowing degradation of IgG in proportion to its plasma concentration (65). If IVIG

depleted of anti-aGal is repeatedly infused, the level of anti-aGal should steadily fall.

Our preliminary data using IVIG (depleted of anti-a Gal) in experiments involving the

i.v. infusion of porcine peripheral blood progenitor cells into baboons indicate that

significantly greater hematopoietic cell chimerism can be obtained than when IVIG is

not administered (see Section II, Chapter 2). We believe this beneficial effect is

brought about, at least in part, by inhibition of macrophage function.

Intravenous Infusion of Oligosaccbarides

Yet another approach to 'neutralize' anti-aGal is to administer a large quantity of

soluble antigen, thus blocking anti-aGal in vivo. It has been demonstrated that many

oligo saccharides terminating in aGalal can inhibit the action of anti-aGal (4-

6,57,66-68). Ye et aI. (57) demonstrated that the i.v. infusion of melibiose and/or

arabinogalactan in a very high concentration was effective in (partially) eliminating

cytotoxicity of the serum to PKI5 cells in baboons. Infusion of such high

concentrations, however, resulted in severe toxicity. More recently, i.v. administration

of different oligosaccharides has proved non-toxic and also inhibited anti-a Gal

activity. HAR was delayed, but not abolished (67,68).

METHODS OF SUPPRESSION OF PRODUCTION OF ANTI-aGAL

ANTIBODIES

Antibodies are produced by B lymphocytes and plasma cells. If it were possible to

suppress selectively the cells responsible for the production of anti-aGal, a major step

would have been taken in achieving long-term xenograft acceptance. As this is not yet

68


a reality, present studies at our center are directed towards temporarily destroying or

suppressing all B cells andlor plasma cells by means of (i) irradiation, (ii)

conventional or novel phannacologic agents, (iii) anti-B cell or plasma cell

monoclonal antibodies, or (iv) immunotoxins.

Whole Body Irradiation (WBI)

Irradiation causes death preferentially of rapidly dividing cells, such as malignant

cells and cells involved in hematopoiesis, and is currently being used as an element in

the conditioning regimen for bone marrow transplantation (69). It has therefore been

included in some protocols aimed at achieving mixed hematopoeitic chimerism and

the induction of tolerance (39,49), WBI of 300cGy (which is a non-lethal dose from

which the bone marrow can spontaneously recover) leads to the temporary ablation of

B cells (but not plasma cells (70)) (Figs. 2A & 2B), but is associated with only a small

and temporary reduction in the production of antibody, including anti-aGal

(38,39,49,70). B cell recovery is fairly rapid (Figure 2C). WBI, therefore, although

creating "space" in the bone marrow to facilitate engraftment of transplanted

hematopoietic cells, has only a minimal and transient effect on antibody production.

Pharmacologic Agents

Phannacologic immunosuppressive therapy combined with EIA in the depletion and

suppression of production of anti-a Gal has been investigated by Lambrigts et al. (72).

Without immunosuppressive therapy, anti-aGal returned to pre-EIA levels within 48

hours after the last of 3 consecutive EIA procedures. Various combinations of agents

were administered in an effort to slow the rate of recovery of anti-a Gal, but none

suppressed anti-a Gal production to a level of clinical relevance. However, rebound of

anti-aGal to levels greater than or equal to pre-treatment levels was successfully

prevented. Of the agents investigated, mycophenolate mofetil was judged to provide

most suppression of antibody production in the absence of toxic side effects. We have

continued this investigation in baboons by assessing the efficacy of other agents, such

as zidovudine, methotrexate and cladribine (see below). Some agents are worthy of

further discussion.

69

Section //I - Chapter 3

A B c

" ,~ t

• V " :t

Figure 2.

Photomicrographs of baboon LNs at various timepoints pre and post WBI (300 cGy)

immunohistochemically stained with the mouse anti-human monoclonal antibody oceD20.

A. LN before WBI showing nonnal distribution ofB cells in the follic les.

B. LN 5 days after WBJ. Note the near complete depletion of B cells .

c. LN 15 days after WBI. Recovery ofB cells can be seen, beginning at the periphery of the follicles.

Cyclophosphamide

Cyclophosphamide, an alkylating metabolite, has been associated with suppression of

both T and B lymphocytes. lts administration in large doses results in a significant

reduction in the rate of return of anti-a Gal after EIA (72). White and coworkers have

used cyclophosphamide extensively, along with cyclosporine and steroids, in their

hDAF transgenic pig-to-primate heart and kidney transplant models (36,37), Its use

has been associated with significant prolongation of xenograft function for up to

several weeks, although A VR remained difficult to prevent. It is a difficult drug to use

long-term as it can result in severe leukopenia and other side effects which contribute

to morbidity.

70


Mycophenolate mofeti! (MMF)

MMF is an inhibitor of purine synthesis that inhibits T and B cell proliferation and

antibody production. It reduced anti-a Gal return after EIA to a comparable extent to

that obtained by cyclophosphamide, when this latter drug was given in moderate and

safe dosage (72). Minanov et al. (73) described modestly prolonged graft survival of

pig-to-newborn heart transplants treated with a combination of MMF, cyclosporine,

and methylprednisolone (from 3.6 to 6.2 days).

Melphalan

This alkylating agent has been used as mainstay treatment for multiple myeloma,

often in combination with steroids. It would appear to be a promising drug for use in

xenotransplantation in view of its effect on plasma cells, which are almost certainly

the major source of anti-aGal production. However, Lambrigts et al. found it rather

less effective in depleting anti-a Gal than cyclophosphamide or MMF (72), although

there would appear to be a delayed effect on antibody production that may be

important. Our impression is that prolonged therapy with melphalan (over several

weeks) combined with intermittent courses of EIA may slowly reduce the number of

anti-a Gal antibody-producing cells.

Zidovudine (AZT)

Zidovudine is a purine analogue used in the treatment of HIV infection (74). It

inhibits viral reverse transcriptase, but also inhibits the manunalian DNA-polymerase

that is responsible for DNA synthesis of mitochondria. Our hypothesis was that, since

antibody production by B and plasma cells is a highly energy-dependant process,

depletion of cellular mitochondria might prevent antibody from being produced. We

have administered zidovudine to baboons while depleting anti-a Gal by EIA. There

was significant reduction of anti-aGal, particularly of IgG, suggesting some

suppressive effect of the drug (Fig. 3). However, this reduction would not have been

sufficient by itself to be of clinical impact.

71


Zidovudine 1200 mg/day OayOto28

Figure 3.

Graph depicting the anti-a Gal IgG and JgM profiles in a naIve baboon receiving zidovudine from day 0

through day 28. EIA was performed on the days indicated by short arrows. Following successful

removal of anti-aGal by repeated ElA, the rate of return of antibody was slow, but return to pre

treatment level was not significantly prevented.

A1ethotrexate

Methotrexate is a dihydrofolate reductase inhibitor, and is another drug that has been

used in the treatment of malignant lymphoid tumors. It has also been used as

adjunctive therapy following organ transplantation in patients with steroid-resistant or

recurrent acute rejection (75). We have assessed its effect on anti-cx.Gal production in

baboons. Although it had no apparent myelosuppressive effect (in contrast to its effect

in humans), anti-aGallevels remained low after EIA for more than 2 weeks.

Cladribine (Leustatin)

Cladribine (leustatin) IS a novel antineoplastic drug used for therapy III

lymphoproliferative malignancies. It is being explored in experimental transplantation

72


as an immunosuppressive agent in both small and large animal models (76). Unlike

other chemotherapeutic agents affecting purine metabolism, it is not only toxic to

actively dividing lymphocytes but also to quiescent lymphocytes and monocytes, in

which it induces apoptosis. In our studies, its efficacy as a suppressor of antibody

production is at least equal to that of most of the other agents tested. Following EIA,

baboons receiving c1adribine maintained a decrease in anti-aGal levels of

approximately 50% - 80% for approximately 2 weeks.

Anti-B Cell Monoclonal Antibodies (mAbs)

B cells can be targeted with mAbs directed against B cell-specific surface antigens.

Several such mAbs have proven successful in reducing the mass of B cell lymphomas

in in vitro, preclinical, and clinical studies (76-81). Following coating by mAb, the

lymphoma cells are cleared mainly through antibody-dependant cell-mediated

cytotoxicity. Such mAbs may prove useful in depleting nonnal B cells and thus

perhaps lead to reduced antibody production. CD20, expressed on the surface of

normal B cells, is targeted by the human-mouse chimeric antibody IDEC-C2B8. This

mAb is effective in depleting malignant B cells in the blood of patients with B cell

lymphoma; tumor regression has been documented (77-81). In our laboratory,

baboons have been treated with a 4 week course (xl weekly) of anti-CD20 mAb, after

which no B cells could be detected in the blood or bone marrow for up to 3 months.

Lymph nodes showed an 80% decrease in B cells 5 weeks after the initial dose, with

recovery beginning by week 6. After a course of EIAs, anti-a Gal levels remained

reduced for 3-4 weeks but the reduction was relatively modest when compared with

the extent of B cell deletion.

Immunotoxins

Some anti-B cell mAbs can be conjugated to toxins, such as ricin A or sapporin, and

become more efficient in depleting B cells (82,83). CD22 is involved with the

generation of mature B cells within the bone marrow, blood, and marginal zoneS of

lymphoid tissues. An anti-CD22 mAb has been successful in the treatment of non

Hodgkin lymphoma (84). An anti-CD22 mAb was conjugated to the ricin A chain

(kindly provided by Ellen Vitetta) and administered to a baboon. Its administration

73

Section Ill- Chapter 3

led to a rapid reduction of B cells in the blood, bone marrow and lymph nodes, but its

prolonged effect was impaired by the development of anti-murine and anti-ricin

antibodies. It was therefore difficult to assess its true effect on anti-aGal antibody

levels but, even with impairment of its efficacy by the development of antibodies, its

immediate effect looked encouraging.

Prevention of Induced Antibodies

Induced anti-aGal IgG, and possibly antibody directed against new porcine (non-aGal)

antigenic determinants, are considered to playa major role in A VR. These newly

synthesized antibodies are ahnost certainly produced by a T cell-dependent mechanism.

Prevention of this induced reponse may therefore prove to be a major step in the

prevention of A YR. The costimulatory pathway of CD40 and the T cell ligand, CD40L

(or CD154), is crucial for effective activation of T cells to antigen (85) and plays an

important role in establishing T cell-dependent B cell activity (86). Blockade of this

pathway alone or in combination with blockade of the B7/CD28 pathway effectively

prolongs survival of skin and organ allografts in rodents (87) and of kidney allografts in

monkeys (88). At our center, costimulatory blockade has been shown to facilitate the

establishment of mixed chimerism and tolerance to skin allografts in rodents when

combined with a nonmyeloablative regimen (89). In xenotransplantation, costimulatory

blockade has allowed prolonged survival of rat-to-mouse skin and heart grafts, as well as

pig-to-mouse skin grafts (90). Recently, we have incorporated murine anti-human

CD40L mAb therapy in a regimen aimed towards the induction of mixed hematopoietic

chimerism in baboons (91). Without anti-CD40L mAb, high doses of porcine blood

progenitor cells transplanted into baboons lead to sensitization to aGal (with a IOO-fold

increase in anti-aGal IgG) and the development of antibody to new (non-aGal) epitopes

Fig. 4).

"When anti-CD40L mAb was added to the regimen, sensitization to all pig antigens,

including aGal, was prevented (Fig. 5). Although there was a return of aGal-reactive

IgM and IgG to pretransplant levels, there was no increase of either immunoglobulin

above those levels. The development of antibody to other pig antigens was also

74


prevented. These data suggest that anti-CD40L rnAb may be of great value in preventing

A VR in pig-to-primate xenotransplantation.

CONCLUSIONS I FUTURE INVESTIGATIONS

Anti-aGal plays a major role in complementMmediated HAR of a porcine vascularized

organ. If this is avoi.ded, the development of A VR would appear to be related to

induced high affinity antiMaGal IgG and antibody to non-aGal porcine detenninants.

Unless accommodation develops, or tolerance is achieved during a window when

there is no antibody being produced, it would appear to be necessary to suppress anti

aGal production permanently, which would clearly be difficult, if not impossible. If

tolerance is the goal, it would at least appear to be necessary to suppress anti-aGal

production for a period of time sufficient to allow for apoptosis of aGal-reactive

B/plasma cells by the presence of pig cells expressing the aGal epitope.

Several strategies have been developed for depletion and maintenance of depletion of

anti-aGal. EIA with an aGalMspecific immunoaffinity column is currently effective in

depleting anti-aGal, of both IgG and IgM subclasses. Our experience with almost 300

EIAs in baboons indicates that a course of 3 EIAs results in 99% and 97% reductions in

IgG and IgM. respectively. Unfortunately, depletion cannot be maintained without

repeated EIA.

The combination of EIA with agents such as cyclophosphamide, :MM:F, zidovudine,

metphalan, and cladribine have contributed to a reduced rate of return of anti-a Gal, but

not to the extent to be clinically relevant. In the absence of an effective anti-plasma cell

rnAb, current attention is directed towards B cells. The normal life-span of human

plasma cells remains uncertain but, if B cells could be eliminated, no new plasma cells

would be produced. Following the death of existing plasma cells, a state of

hypogammaglobulinemia might be reached. During this period, newly-developing aGal

reactive B cells could be tolerized by successful porcine hematopoietic cell

75

:J' E 0, 2, .0

"" " CJ

~ c

""

Section lll- Chapter 3

transplantation. Although this temporary state of hypogammaglobulinemia would not be

without risk to the subject, steps could be taken to limit this risk.

100001 -0- IgG __ lgM

1000

1000

100

10

I

I

! 100

10

·20 20 40 60 80 100 120 140

Time (Days)

1 Pre Day 20

c:::=J None Adsorbed p///J MatrixAdsorbed ~ Pig Cell Adsorbed

Figure 4.

Anti-aGal IgG and IgM (left) and non-aGal-reactive antibody (right) responses as median fluorescence

intensity (MFI) following porcine peripheral blood progenitor cell (PBPC) transplantation in

representative baboons receiving a tolerance inducing regimen without anti-CD40L mAb. The arrows

(left) indicate the first day of porcine PBPC transplantation, which was administered after the third and

final EIA (day 0). In (right), column 1 represents the anti-pig antibody level, column 2 represents the

same serum after immunoadsorption of anti-aGal antibody, and column 3 represents this serum

depleted of both aGal-reactive and non-a Gal-reactive antibodies. The difference between columns 1

and 2 therefore indicates the amount of anti-a Gal antibody, and the difference between columns 2 and

3 indicates the amount of anti-nona Gal antibody.

Left. A rise in both anti-aGal IgG and IgM occurred by day 10, indicating sensitization

to the Gal detenninants on the PBPe.

Right. Antibody directed to porcine nonaGal detenninants on the PBPC developed within

20 days.

76

10000

1000

:?

~ ""

100

.c « ro

'" 10 9 :g «

01

-20

_____ IgG

_lgM

:t---r~ ~

20 40 60

Time (Days)

Figure 5.

1000

100

u:: :;;

10

1

-,


Pre Day 20

c:J None Adsorbed ~ Matrix Adsorbed ~ Pig Cell Adsorbed

Anti-aGal IgG and IgM (left) and non-aGal-reactive antibody (right) responses as meOian I1uorescence

intensity (MFI) following porcine peripheral blood progenitor cell (PBPC) transplantation in

representative baboons receiving a tolerance inducing regimen with anti-CD40L mAb.

Left. No rise in anti-aGal IgG or IgM over baseline occurred.

Right. Antibody directed to porcine nonaGal determinants on the PBPC did not develop

With regard to current progress, however, the ability of anti-CD40L mAb to prevent the

induced antibody response to pig cells represents a significant step in overcoming the

immunologic barriers to xenotransplantation, and will undoubtedly facilitate the survival

of transplanted pig organs in primates and the induction of tolerance.

REFERENCES

I. Perper RJ, Najarian JS. Experimental renal heterotransplantation. L In widely divergent species. Transplantation 1966; 4; 377

77


2. Galili U, Shohet SB, Kobrin E, et al. Man, apes, and Old World monkeys differ from other mammals in the expression of a~galactosyl epitopes on nucleated cells. 1 BioI Chern 1988; 263: 17755

3. Galili U, Rachmilewitz EA, Peleg A, et al. A unique natural human IgG antibody with anti-agalactosyl specificity. 1 Exp Med 1984; 260: 1519

4. Good AH, Cooper DKC, Malcolm Al, et al. Identification of carbohydrate structures that bind human anti-porcine antibodies: implications for discordant xenografting in humans. Transplant Proc 1992; 24: 559

5. Cooper DKC, Good AH, Koren E, et al. Identification of a-galatosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies: relevance to discordant xenografting in man. Transplant Immunol 1993; 1: 198

6. Cooper DKC, Koren E, Oriol R. Oligosaccharides and discordant xenotransplantation. Immunol Rev 1994; 141: 31

7. Koren E, Neethling FA, Richards S, et al. Binding and specificity of major immunoglobulin classes ofprefonned human anti-pig heart antibodies. Transplant Int. 1993; 6: 351

8. Kujundzic M, Koren E, Neethling FA, et al. Variability of anti~aGal antibodies in human serum and their relation to serum cytotoxicity against pig cells. Xenotransplantation 1994; I: 58

9. Galili U, Mandrell RE, Hamadeh RM, et al. The interaction between the human anti-a~galactosyl IgG (anti-Gal) and bacteria of the human flora. Infect Immun 1988; 57: 1730

10. Sandrin MS, Vaughan HA, Dabkowski PL, et aJ. Anti-pig 19M antibodies in human serum react predominantly with Oala(J~3)Gal epitopes. Proc Natl Acad Sci USA 1993; 90: 11391

11. Galili U. Interaction of the natural anti-Gal antibody with agalactosyl epitopes: a major obstacle for xenotransplantation in humans. Immunol Today 1993; 14: 480

12. Galili U. Anti-agalactosyl (anti-Gal) antibody damage beyond hyperacute rejection. In: Cooper DKC, Kemp E, Platt lL, \Vhite DlG (eds). Xenotransplantation, 2nd ed. Heidelberg: Springer 1997. p 95

13. Bach FR, Robson SC, Winkler H, et al. Barriers to xenotransplantation. Nat Med 1995; 1: 869 14. Oriol R, Ye Y, Koren E, et al. Carbohydrate antigens of pig tissues reacting with human natural

antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation. Transplantation 1993; 56: 1433

15. Oriol R, Barthold F, Bergemer A~M, et a1. Monomorphic and polymorphic carbohydrate antigens on pig tissues: Implications for organ xenotransplantation in the pig~to-man model. Transplant lnt 1994;7:405

16. Cooper DKC. Xenoantigens and xenoantibodies. Xenou'ansplantation 1998; 5: 6 17. McMorrow 1M, Comrack CA, Nazarey PP, et al. Relationship between ABO bloodgroup and

levels of Gal alpha,3galactose-reactive human immunoglobulin G. Transplantation 1997; 64: 546 18. Parker W, Lundberg-Swanson KL, Holzknecht ZE, et a!. Isohemagglutinins and xenoreactive

antibodies: members of a distinct family of natural antibodies. Hum lmmunol 1996; 45: 94 19. Platt lL, Fischel RJ, Matas Al, et al. Immunopathology of hyperacute xenograft rejection in a

swine-to-primate model. Transplantation 1991; 52: 514 20. Platt lL, Vercelotti OM, Lindman BJ, et al. Release of heparan sulfate from endothelial cells:

Implications for pathogenesis of hyperacute rejection. J Exp Med 1990; 171: 1363 21. Koren E, Kujundzic M, Koscec M, et a1. Cytotoxic effects of human preformed anti-Gal IgG and

complement on cultured pig cells. Transplant Proc 1994; 26: 1336 22. Joziasse DH, Shaper lR, labs EW, et al. Characterization of an al,3-galactosyltransferase

homologue on human chromosome 12 that is organized as a processed pseudogene. J BioI Chem 1991; 266: 6991

24.

25.

78

Oalili U, Andrews P. Suppression of a~galactos)l epitopes synthesis and production of the natural anti-Gal antibody: a major evolutionary event in ancestral Old World primates. 1 Hum Evo11995; 29:433 Lambrigts 0, Sachs DH, Cooper DKC. Discordant organ xenotransplantation in primates. World experience and current status. Tranplantation 1998; 66: 547 Rose AG, Cooper DKC, Human PA, et aJ. Histopathology of hyperacute rejection of the heart: experimental and clinical observations in allografts and xenografts. J Heart Lung Transplant 1991; \0; 223


26. Cooper DKC, Human PA, Lexer G, et a1. Effects of cyc1osporine and antibody adsorption on pig cardiac xenograft survival in the baboon. J Heart Lung Transplant 1988; 7: 238

27. Alexandre GPJ, Giane!!o P, Latinne D, et al. Plasmapheresis and splenectomy in experimental renal xenotransplantation. In: Hardy MA (ed). Xenograft 25. New York, Elsevier, 1989. p 28.

28. SabJinski T, Latinne D, Gianello P, et a!. Xenotransplantation of pig kidneys to nonhuman primates: I. Development of the model. Xenotransplantation. 1995; 2: 264

29. Cooper DKC, Cairns TDH, Taube DH. Extracorporeal immunoadsorption of anti-pig antibody in pigs using aGal oligosaccharide immunoaffinity columns. Xeno 1996; 4: 27

30. Kaplon RJ, Michler RE, Xu H, et al. Absence of hyperacute rejection in newborn pig-to-baboon cardiac xenografts. Transplantation 1994; 59: 1

31. Collins BH, Cotterell AH, McCurry KR, et al. Cardiac xenografts between primate species provide evidence for the importance of the a-galactosyl determinant in hyperacute rejection. J Immunol 1995; 154;5500

32. Leventhal JR, Dalmasso AP, Cromwell JW, et aL Prolongation of cardiac xenograft survival by depletion of complement. Transplantation. 1993; 55: 857

33. Kobayashi T, Taniguchi S, Neethling FA, et aL Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy. Transplantation 1997; 64: 1255

34. Cozzi E, White DJG. The generation of transgenic pigs as potential organ donors for humans. Nat Med 1995; I: 964

35. McCurry KR, Kooyman DL, Alvarado CG, et a!. Human complement regulatory proteins protect swine to primate cardiac xenografts from human injury. Nat Med 1995: 1; 123

36. Schmoeckel M, Bhatti FN, Zaidi A, et al. Orthotopic heart transplantation in a transgenic pig-toprimate model. Transplantation 1998; 65: 1570

37. Zaidi A, Schmoeckel M, Bhatti F, et al. Life-supporting pig-to-primate renal xenotransplantation using genetically modified donors. Transplantation 1998; 65: 1584

38. Kozlowski T, Monroy R, Xu, Y, et al. Anti-Galal-3Gal antibody response to porcine bone marrow in unmodified baboons and baboons conditioned for tolerance induction. Transplantation 1998; 66; 176

39. Kozlowski T, Shimizu A, Lambrigts 0, et al. Porcine kidney and heart transplantation in baboons undergoing a tolerance inducing regimen and antibody adsorption. Transplantation 1999; 67: 18

40. Groth CO, Korgsen 0, Tibell A, et al. Transplantation of fetal porcine pancreas to diabetic patients: Biochemical and histological evidence for graft survival. Lancet 1994; 344: 1402

41. Galili U, Latemple DC, Walgenbach AW, et aL Porcine and bovine cartilage transplants in cynomolgus monkeys: II. Changes in anti-gal response during chronic rejection. Transplantation 1997;63;646

42. Goodman DL, Millan MT, Ferran C, et al. Mechanisms of delayed xenograft rejection. In: Cooper DKC, Kemp E, Platt JL, White DJG (eds). Xenotransplantation, 2nd ed. Heidelberg,Springer, 1997. p77

43. Buhler L, Friedman T, Iacomini J, Cooper DKC. Xenotransplantation - state of the art ~ update 1999. Front Bioscl 1999; 4: D416

44. Chopek MW, Simmons RL, Platt J1. ABO incompatible renal transplantation: initial immunopathologic evaluation. Transplant Proc 1987; 19: 4553

45. Alexandre GPJ, Squifflet JP, De Bruyere M, et aL Present experiences in a series of 26 ABOincompatible living donor renal allografts. Transplant Proc 1987; 19: 4538

46. Palmer A, Welsh K, Gjorstrup P, et al. Removal of HLA antibodies by extracorporeal immunoadsorption to enable renal transplantation. Lancet 1989; 1: 10

47. Bach FH, Platt JL, Cooper DKC. Accommodation - the role of natural antibody and complement in discordant xenograft rejection. In: Cooper DKC, Kemp E, Reemtsma K, White DJG (eds). Xenotransplantation, 1st ed. Heidelberg, Springer, 1991. P 81

48. Bach FH, Ferran C, Hechenleitner P, et a1. Accomodation of vascularized xenografts: expression of "protective genes" by donor endothelial cells in a host ThZ cytokine environment. Nat Med 1997; 3; 196

49. Sykes M, Sachs DR Xenogeneic tolerance through hematopoietic eel! and thymic transplantation. In: Cooper DKC, Kemp E, Platt JL, White DJG (eds). Xenotransplantation, 2nd ed. Heidelberg, Springer,: 1997. p 496

79

Section I/l- Chapter 3

50. Moberg A W, Shons AR, Gerwurz H, et al. Prolongation of renal xenografts by the simultaneous sequestration of prefonned antibody, inhibition of complement, coagulation, and antibody synthesis. Transplant Proc 1971; 3: 538

51. Slapak M, Greenbaum M, Bardawil W, et al. Effect of heparin, arvin, liver perfusion, and heterologous antiplatelet serum on rejection of pig kidney to dog. Transplant Proc 1971; 3: 558

52. Shons AR, Beir M, Jetzer J, et al. Techniques of in vivo plasma modification for the treatment of hyperacute rejection. Surgery 1973; 73: 28

53. Tennan DS, Garcia-Rinaldi R, McCalmon R, et al. Modification of hyperacute renal xenograft rejection after extracorporea! immunoadsorption of heterospecific antibody. Int J Artif Organs 1979; 2: 35

54. Lexer G, Cooper DKC, Rose AG, et al. Hyperacute rejection in a discordant (pig-to-baboon) cardiac xenograft model. J Heart Transplant 1986; 5: 411

55. Azimzadeh A, Meyer C, Watier H, et al. Removal of primate xenoreactive natural antibodies by extracorporeal perfusion of pig kidneys and livers. Transpl Immunol 1998; I: 13

56. Macchiarini P, Oriol R, Azimzadeh A, et a1. Evidence of human non-alpha-galactosyl antibodies involved in the hyperacute rejection of pig lungs and their removal by pig organ perfusion. J Thorac Cardiovasc Surg 1998; 5: 831

57. Ye Y, Neethling FA, Niekrasz M, et a1. Evidence that intravenously administered alpha-galactosyl carbohydrates reduce baboon serum cytotoxicity to pig kidney cells (PKI5) and transplanted pig hearts. Transplantation 1994; 58: 330

58. Taniguchi S, Neethling FA, Korchagina EY, et al. In vivo immunoadsorption of anti pig antibodies in baboons using a specific Galal-3Gal column. Transplantation 1996; 10: 1379

59. Xu Y, Lorf T, Sablinski T, et al. Removal of anti-porcine natural antibodies from huma and nonhuman primate plasma in vitro and in vivo by a Galal-3Galpl-4Glc-X immunoaffinity column. Transplantation 1998; 65: 172

60. Kozlowski T, Ierino FL, Lambrigts D, et a1. Depletion ofanti-aGall-3Gal Antibody in baboons by specific immunoaffinity columns. Xenotransplantation 1998; 5: 122

61. Koren E, Milotic F, Neethling FA, et a1. Monoclonal antiidiotypic antibodies neutralize cytotoxic effects of anti-aGal antibodies. Transplantation 1996; 62: 837

62. Wordell CJ. Use of intravenous inunune globulin therapy: an overview. DICP 1991; 25: 805 63. Jordan SC, Tyan D, Czer L, et al. Immunomodulatory actions of intravenous immunoglobulin

(IVIG): potential applications in solid organ transplant recipients. Pediatr Transplant 1998; 2: 92 64. Magee JC, Collins BH, Harland RC, et al. Immunoglobulin prevents complement mediated

hyperacute rejection in swine-to-primate xenotransplantation. J Clin Invest 1995; 96: 2404 65. Yu Z, Lennon VA. Mechanism of intravenous immune globulin therapy in antibody-mediated

autoimmune diseases. N Engl J Med 1999; 340: 227 66. Neethling FA, Joziasse 0, Bovin N, et al. The reducing end of aGal oligosaccharides contributes

to their efficiency in blocking natural antibodies of human and baboon sera. Transplant Int. 1996; 9:98

67. Simon PM, Neethling FA, Taniguchi S, et al. Intravenous infusion of aGal oligosaccharides in baboons delays hyperacute rejection of porcine heart xenografts. Transplantation. 1998; 63: 346

68. Romano E, Neethling FA, Nilsson KG!, et a1. Intravenous synthetic aGal saccharides delays hyperacute rejection following pig-to-baboon heart transplantation. Xenotransplantation. In press.

69. Lindsley K, Deeg HJ. Total body irradiation for marrow or stem-cell transplantation. Cancer Invest 1998; 16: 424

70. Kimikawa M, Kawai T, Sachs DH, et a!. Mixed chimerism and transplantation tolerance induced by a nonlethal preparative regimen in cynomolgus monkeys. Transplant Proc 1997; 29: 1218

71. Clave E, Socie G, Cosset JM. Multicolor flow cytometry analysis of blood cell subsets in patients given total body irradiation before bone marrow transplantation. lnt J Radiat Oncol Bioi Phys 1995; 34: 881

72. Lambrigts D, Van Calster P, Xu Y, et al. Phannacologic immunosuppressive therapy and extracorporeal immunoadsorption in the suppression of anti-a Gal antibody in the baboon. Xenotransp!antation 1998; 5: 274

73. Minanov OP, Artrip JH, Szabolcs M, et al. Triple immunosuppression reduces mononuclear cell infiltration and prolongs graft life in pig-to-newborn baboon cardiac xenotransplantation. J Thorac Cardiovasc Surg 1998; 115: 998

74. Lindback S, Vizzard J, Cooper DA, et al. Long-tenn prognosis following zidovudine monotherapy

80


in primary human immunodeficiency virus type I infection. J Infect Dis 1999; 179: 1549 75. Costanzo-Nordin M, Grusk S, Silver M, et al. Reversal of recalcitrant cardiac allograft rejection

with methotrexate. Circulation 1988; 78:III 47 76. Gorski A. Grieb P, Korczak-Kowalska G. et al. Cladribine (2-chloro-deoxyadenosine, CDA): an

inhibitor of human Sand T cell activation in vitro. Immunophannacology 1993; 26: 197 77. Reff ME, Carner K, Chambers KS, et a1. Depletion of B cells in vivo by a chimeric mouse human

monoclonal antibody to CD20. Blood 1994; 83: 435 78. Maloney DG, Grillo-Lopez AJ, Bodkin DJ, et al. IDEC-C2B8: results of a phase I multiple-dose

trial in patients with relapsed non-Hodgkin's lymphoma. J Gin Oncoll997; 15: 3266 79. Tobinai K, Kobayashi Y, Narabayashi M. et al. Feasibility and phannacokinitic study of a

chimeric anti~CD20 monoclonal antibody (lDEC-C2B8) in relapsed B cell lymphoma. The lDECC2B8 study group. Ann Oncol 1998; 9: 527

80. Coiffier B, Haioun C, Ketterer N, et al. Rituximab (anti-C020 monoclonal antibody) for the treatment of patients with relapsing or refractory aggressive lymphoma: a multicenter phase II study. Blood 1998; 92: 1927

81. McLaughlin P, Grillo-Lopez AJ, Link BK, et al. Rituximab chimeric anti~CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four dose treatment program. J Clin Oncol 1998; 16: 2825

82. Bolognessi A, Tazzari PL, Olivieri F, et a1. Evaluation of immunotoxin containing single-chain ribosome-inactivating proteins and an anti-CD22 monoclonal antibody (OMI24): in vitro and in vivo studies. Sr J Haematol 1998:. 10 I: 179

83. Sausville EA, Headlee D, Stetler-Stevenson M, et a!. Continuous infusion of the anti-C022 immunotoxin IgG-RFB4-SMPT-dgA in patients with B cell lymphoma: a phase [ study. Blood 1995;85; 3457

84. Tedder TF, Tuscano J. Sato S, et al. CD22, a B lymphocyte -specific adhesion molecule that regulates antigen receptor signaling. Annu Rev Immunol 1997; 15: 481

85. Armitage RJ, Fanslow WC, Strockbine L, et a!. Molecular and biological characterization ofa murine ligand for CD40. Nature 1992; 357: 80

86. Noelle R, Roy M, Shepherd D, et aJ. A 39-kDa protein on activated helper T cells binds CD40and transduces the signal for cognate activation of B cells. Proc Natl Acad Sci USA 1992; 89: 6550

87. Larsen CP, Elwood ET, Alexander DZ, et al. Long-tenn acceptance of skin and cardiac allografts after blocking CD40 ligand. Nature 1996; 381: 434

88. Kirk AD, Harlan DM, Annstrong NN, et al. CTLA4-lg and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci USA 1997; 94: 8789

89. Wekerle T, Sayegh MH, Hill J, et a1. Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance. J Exp Med 1998; 187: 2037

90. Elwood ET, Larsen CP, Cho HR, et al. Prolonged acceptance of concordant and discordant xenografts with combined CD40 and CD28 pathway blockade. Transplantation 1998; 65: 1422

91.Buhler L, Awwad M, Basker M, et al. A nonmyeloablative regimen with CD40L blockade leads to humoral and cellular hyporesponsiveness to pig hematopoietic cells in baboons. Transplant Proc. 2000: 32; 1100.

81


82

4 Effects of specific anti-B and/or anti-plasma cell immunotherapy on xenoreactive antibody production in baboons

Adaptedfrom: Alwayn IPJ', Xu y', Basker M', Wu C, Buhler L, Lambrigts D, Treter 5, Harper D, Kitamura H. Vitetta E, Abraham S, Awwad M, White-Scharf ME, Sachs DH, Thall A, and Cooper DKC. Effects of specific anti-B and/or anti-plasma cell immunotherapy on antibody production in baboons: depletion of CD20- and CD22-positive B cells does not result in significant decreased production of anti-aGa! antibody. Xenotransplantation 2001. In press. & Basker M, Alwayn IPJ, Treter S, Harper D, Buhler L, Andrews D, Thall A, Lambrigts D, Awwad M, White-Scharf M, Sachs DB, and Cooper DKC. Effects of B cell/plasma cell depletion or suppression on anti-Gal antibody in the baboon. Transpl. Proc. 2000;32(5): 1009. 'Authors contributed equally.

PUBMED-ID

HYPERLINK "/pubmed/10936323"Effect of B cell/plasma cell depletion or suppression on anti-Gal antibody in the baboon. Basker M, Alwayn IP, Treter S, Harper D, Buhler L, Andrews D, Thall A, Lambrigts D, Awwad M, White-Scharf M, Sachs DH, Cooper DK. Transplant Proc. 2000 Aug;32(5):1009. No abstract available. PMID: 10936323 [PubMed - indexed for MEDLINE]

PUBMED-ID

HYPERLINK "/pubmed/10936323"Effect of B cell/plasma cell depletion or suppression on anti-Gal antibody in the baboon. Basker M, Alwayn IP, Treter S, Harper D, Buhler L, Andrews D, Thall A, Lambrigts D, Awwad M, White-Scharf M, Sachs DH, Cooper DK. Transplant Proc. 2000 Aug;32(5):1009. No abstract available. PMID: 10936323 [PubMed - indexed for MEDLINE]

Section 1/1- Chapter 4

ABSTRACT

Background. Anti-Galal-3Gal antibodies (anti-aGal Ab) are a major barrier to

clinical xenotransplantation as they are believed to initiate both hyperacute and acute

humoral rejection. Extracorporeal immunoadsorption (EIA) with aGal

oligosaccharide columns temporarily depletes anti-aGal Ab, but their return is

ultimately associated with graft destruction. We, therefore, assessed the ability of two

immunotoxins (IT) and two monoclonal antibodies (mAb) to deplete B and/or plasma

cells both in vivo and in vitro in baboons, and to observe the rate of return of anti

aGal Ab following EIA.

Methods. The effects of the mouse anti·human IT anti-CD22·ricin A (ocCD22·IT.

directed against a B cell determinant) and anti·CD38-ricin A (ocCD38-IT. Band

plasma cell determinant) and the mouse anti-human anti-CD38 mAb (ocCD38 mAb)

and mouselhuman chimeric anti-human anti-CD20 mAb (ocCD20 mAb. Rituximab. B

cell determinant) on B and plasma cell depletion and anti-aGal Ab production were

assessed in vivo in baboons (n=9) that had previously undergone splenectomy. For

comparison, two baboons received nonmyeloablative whole body irradiation (WBI)

(300 cGy). and one received myeloablative WBI (900 cGy). Five baboons were

administered either ocCD22-IT (at 0.125 - 0.19 mg/kg x 4 doses; n~2) or ocCD38·IT

(0.1 - 0.6 mg/kg x 14 doses; n~3). One baboon received xCD38 mAb at OJ mg/kg x

21 doses. Three baboons received ocCD20 mAb at 20 mg/kg x 4 doses, either with

(n~l) or without (n~2) WBI (150 cGY). Depletion ofB cells was monitored by flow

cytometry of blood, bone marrow (BM) and lymph nodes (LN), and by histology of

LN. EIA was carried out after the therapy and anti-aGal Ab levels were measured

daily. These agents were further analyzed in vitro.

Results. In vivo, WBI (300 or 900 cGy) resulted in 100% B cell depletion in blood

and BM, > 80% depletion in LN, with substantial recovery of B cells after 21 days

and only transient reduction in anti-aGal Ab after EIA. The effect of both murine ITs

was in part limited by the development of ccmurine andlor ocricin antibodies.

However, ocCD22-IT depleted B cells by >97% in blood and BM, and by 60% in LN.

84

Anfi-B cell immunotherapy

but a rebouud of B cells was observed after 14 and 62 days in LN and blood,

respectively. At 7 days, serum anti-aGal IgG and IgM Ab levels were reduced by a

maximum of 40 - 45% followed by a rebouud to levels up to 12-fold that of baseline

anti-aGal Ab by day 83 in one baboon. The results obtained with ocCD38-IT were

inconclusive. This may have been, in part, due to inadequate conjugation of the toxin.

Cell coating was 100% with ocCD38 mAb, but no changes in anti-aGal Ab production

were observed. ocCD20 mAb resulted in 100% depletion of B cells in blood and BM,

and 80% in LN, with recovery of B cells starting at day 42. Adding 150cGy WBI at

this time led to 100% depletion of B cells in the BM and LN. Although B cell

depletion in blood and BM persisted for> 3 months, the reduction of serum anti-a Gal

IgG or IgM Ab levels was not sustained beyond 2 days. In vitro, ocCD22-IT inhibited

protein synthesis in the human Daudi B cell line more effectively than ocCD38-IT.

Upon differentiation of B cells into plasma cells, however, less inhibition of protein

synthesis after ocCD22-IT treatment was observed. Depleting CD20-positive cells in

vitro from a baboon spleen cell population already depleted of granulocytes,

monocytes, and T cells led to a relative enrichment of CD20-negative cells, i.e.,

plasma cells, and consequently resulted in a significant increase in anti-a Gal Ab

production by the remaining cells, whereas depleting CD38-positive cells resulted in a

significant decrease in anti-aGal Ab production.

Conclusions. ocCD20 mAb + WBI totally and efficiently depleted B cells in blood,

BM, and LN for >3 months in vivo, but there was no sustained clinically significant

reduction in serum anti-aGal Ab. The majority of antibody secretors are CD38-

positive cells, but targeting these cells in vitro or in vivo with ocCD38-IT was not very

effective. These observations suggest that B cells are not the major source of anti

aGal Ab production. Future efforts will be directed towards suppression of plasma

cell function.

INTRODUCTION

Xenotransplantation of porcine organs is viewed by many clinicians and investigators

as a possible solution to the increasing shortage of donor organs for transplantation

85


(1,2,3). Although many advances have been made in understanding the immunologic

and inflammatory phenomena associated with xenograft rejection (4,5,6), porcine

vascularized xenografts do not survive beyond a median of approximately one month

in nonhuman primate recipients (7,8). There is clear evidence that primate antibodies

directed against tenninal Galal-3Gal oligosaccharides (anti-aGal Ab) on porcine

vascular endothelium (9,10,11,12) are responsible for complement-mediated

hyperacute rejection of porcine organs (13). Furthennore, even if hyperacute rejection

is averted by depletion of anti-aGal Ab, the use of complement regulatory agents or

of porcine organs transgenic for human complement regulatory proteins, anti-aGal

Ab still plays a major role in a delayed rejection response, known variously as acute

vascular rejection, delayed xenograft rejection, or acute humoral xenograft rejection,

which is possibly complement-independent (14).

Specific depletion of anti-aGal Ab by extracorporeal immunoadsorption (ElA) of

baboon plasma through columns containing synthetic aGal oligosaccharides (15,16)

has resulted in prolonged xenograft survival (17,18,19). However, extended survival

is limited by the return of anti-a Gal Ab and by the development of antibodies directed

against other porcine (non-aGal) determinants (19,20). These develop despite

intensive pharmacologic immunosuppressive therapy. Specific therapies that result in

a sustained depletion of anti-aGal Ab and other antibodies will almost certainly prove

beneficial in overcoming acute vascular rejection.

Since antibodies are produced by B and plasma cells, depleting (or inhibiting the

function of) these cells should result in diminished antibody production. Several

pharmacologic agents targeting B cells were assessed for their ability to suppress

production of anti-aGal Ab without significant success (6,21). Fuelled by the

advances made in the treatment of B cell neoplasias using immunotherapy

(22,23,24,25), the effects of specific anti-B and/or plasma cell immunotherapy on B

cell depletion and anti-aGal Ab production in vivo and in vitro in baboons were

investigated. These effects were compared to those achieved in baboons receiving

myeloablative or nomnyeloablative whole body irradiation (WBI), as this has been

86

Anti-B cell immunotherapy

shown to deplete the majority of B cells in blood, bone marrow (BM), and lymph

nodes (LN) (6) by inducing apoptosis in rapidly dividing cells such as lymphocytes

(26).

We selected deglycosylated (dg) ricin A (RTA) for the construction of specific anti-B

and/or plasma cell immunotoxins (IT). The dgRTA chain inhibits protein synthesis by

destroying 60S ribosomal RNA. dgRTA is not efficiently internalized without the

ricin B chain but, once conjugated to a desired monoclonal antibody (mAb),

internalization can be facilitated and specific cell toxicity achieved. Other criteria

used to select effective ITs include: (i) high affinity of the antibody for antigen; (ii)

limited distribution of the targeting antigen in order to avoid non-specific toxicity;

(iii) inhibition of protein synthesis by the toxin at relatively low concentrations due to

effective internalization and intracellular routing.

Four agents were tested in vivo and/or in vitro in baboons.

I. A mouse anti-human CD22 monoclonal antibody (RFB4) conjugated to dgRTA

(ocCD22-IT) was prepared as described previously (27). This IT can inhibit

protein synthesis and antibody production in vitro and give multi-log depletion of

human B cell lymphomas in vivo (28). CD22 is expressed in the cytoplasm of all

B cells and as surface antigen on mature B cells (29). The CD22 antigen is present

on lymphoplasmacytoid cells and is expressed in most B cell lymphomas; but is

diminished on the fully-differentiated plasma cell (29).

2. The unconjugated mouse anti-human CD38 mAb (ocCD38 mAb, OKTlO,

American Type Culture Collection, Manassas, V A). CD38 is expressed on all pre

B and B cells, plasma cells, and thymocytes and is also present on activated T

cells, natural killer cells, myeloblasts, erythroblasts, and BM cells (30).

3. The ocCD38 mAb was conjugated to dgRTA (ocCD38-IT) as described previously

(27). The purity of the IT was assessed by sodium dodecylsulphate-

87

Section fJ[ - Chapter 4

polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions

(31). The preparation contained approximately 60% unconjugated mAb.

4. A chimeric mouse-human mAb directed at the human CD20 surface antigen

(ocCD20 mAb, Rituximab, kindly provided by IDEC Pharmaceuticals, San Diego,

CAl, CD20 is expressed on both resting and activated B cells, but is down

regulated on plasma cells (32). CD20 may also be expressed on follicular

dendritic cells in germinal centers (33).

MATERIAL AND METHODS

In Vivo Experiments

Animals

Baboons (Papio anubis, n~12), of both sexes and weighing approximately 9 - 15 kg,

were obtained from Biomedical Resources Foundation, Houston, TX. In view of the

shortage and expense of nonhuman primates, five of the 12 baboons had been used

previously (>2 months) for other experiments, but recovery of all study parameters

(i.e., B cell counts and anti-aGal Ab levels) had occurred .. All experiments were

performed in accordance with the Guide for the Care and Use of Laboratory Animals

prepared by the National Academy of Sciences and published by the National

Institutes of Health. Protocols were approved by the Massachusetts General Hospital

Subcommittee on Research Animal Care.

Surgical procedures

Splenectomy, intravenous (iv) and intra-arterial catheterization, and EIA were carried

out under inhalation anesthesia. A splenectomy is routinely performed in Oll baboons

because there is evidence that this leads to prolonged survival of xenografts (34, 35)

and, furthermore, we have found that spleens contain a large number of anti-aGal Ab

secreting cells (Xu Y, et al. unpublished data). Baboons were premedicated with

atropine (0,05 mg/kg im) and ketarnine (10 mg/kg im) prior to surgery and were

intubated and ventilated through a closed circuit with oxygen (l - 2 L/min), nitrous

oxide (2 - 4 Llmin), and isoflurane (0,5 - 3,0 %), Splenectomy was perfonned as

88


described previously (I7). Catheters were inserted in the internal and external jugular

veins and carotid artery. The catheters were tunneled subcutaneously to the back, and

brought out through a jacket and tethering system allowing monitoring of arterial

blood pressure, access to blood withdrawal, and continuous or intermittent iv

administration of fluids and/or drugs. Cefazolin (500 mg iv daily) was given as

prophylaxis against infection.

EXlracorporeal immunoadsorption (EIA) of anti-aGal Ab

EIA was performed using an immunoaffinity column (Alberta Research Council,

Alberta, Canada) containing synthetic aGal trisaccharide type VI as previously

described (19,36). Briefly, using a COBE-Spectra apheresis unit (Gambro BCT

International, Lakewood, CO), 3 plasma volrnnes were immunoadsorbed through an

aGal immunoaffinity column at an average perfusion rate of 20 ml/min.

Anticoagulation was achieved with citrate phosphate dextrose adenine, and

hemodynamic stability maintained by volume replacement +1- phenylephrine

infusion. The anti-aGal Ab-depleted plasma was returned to the baboon. Three

consecutive daily EIAs were performed at the time of optimal B cell and/or plasma

cell depletion.

Whole body irradiation (WBI)

Baboons received nonmyeloablative WEI 2xl50 cGy (n~2) or myeloablative WEI

2x450 cGy (n~l) using a Cobalt 60 teletherapy unit (Therapy Services Inc., Frederick,

MD) without specific anti-B and/or anti-plasma cell immunotherapy. Furthermore,

one baboon received 150 cGy WEI 20 days after treatment with ocCD20 mAb (see

below).

Administration of experimental agents in vivo

Treatment with ITs I mAbs was preceded by the iv infusion of 25 mg

diphenhydramine to prevent potential anaphylactic reactions. Commencing on day 0,

the ITs / mAb were administered as bolus iv infusions to baboons. We selected the

dosages for ocCD22-IT (37) and ocCD20 mAb (38) based on maximum tolerated

89


doses from previous clinical studies. The dose of the ocCD38 mAb and the ocCD38-IT

we had were extrapolated from the maximum tolerated dose used for the ocCD22-IT,

since there was no previous clinical experience with this agent.

ocCD22-IT (n:2) at 0.125 - 0.19 mg/kg every other day x 4 doses.

ocCD38 mAb (n:l) at 0.3 mg/kg/day x 21 doses.

ocCD38-IT (n:3) at 0.1 - 0.6 mg/kg/day x 14 doses.

ocCD20 mAb (Rituximab) (n:3) at 20 mglkg/week x 4 doses.

In Vitro Assays

Tissue and cel! preparation

Baboon spleen cells were surgically obtained by splenectomy as described above. Cells

were harvested in RPMI (Gibco BRL, Grand Island, NY), homogenized, and filtered

sequentially through a flow-mesh FM-IOO (PGL Scientifics, Frederick, MD).

Peripheral blood mononuclear cells were prepared from heparinized peripheral blood

obtained from healthy baboons. BM cells were obtained and prepared as described

previously (39). Cells were additionally purified by separation over a Ficoll density

gradient (Histopaque, Sigma, St.Louis, MO).

B cell enrichment

Red blood cells from cell preparations were lysed with ACK buffer (0.15 M NH4Cl,

10 mM KHC03, 0.1 mM Na2EDTA.2H20). Cells were washed and resuspended in

RPMI containing 10% fetal calf serum and gentamicin (Gibco BRL). The average

total yield per spleen was 3-5 x 109 cells. Monocytes were depleted by adherence at

37'C with 5% CO2 in RPMI with 10% fetal calf serum overnight. T cells were

depleted by E-rosetting using AET- (Sigma) treated sheep red blood cells (Cocalico

Biologicals, Inc., Reamstown, PA) following standard procedures. Granulocytes were

removed during the E-rosetting procedure. Alternatively, T cells were depleted with

anti-CD2 (Leu5b, Becton Dickinson, San Jose, CAl followed by goat anti-mouse IgG

magnetic beads (Dynal, Lake Success, NY). B cells were enriched from an initial

90


average of 25-50% to 80-90%, as was assessed by flow cytometry (described below),

The final preparation contained approximately 10% CD2-positive cells.

In vitro activation / differentiation of plasma cells

B cells (2x106 cells/ml in cell suspension medium) were activated with 0.04%

Pansorbin (fixed Staphylococcus Aureus, Calbiochem-Novabiochem Corp., La Jolla,

CAl and 2.5 ng/ml of IL2 (R&D Systems, Minneapolis, MN) for 2 - 3 days at 37'C

with 5% CO,. Cells were washed and subsequently cultured in 2.5 ng/ml IL-2 and

100 ng/ml IL-IO (R&D Systems) for up to 8 days, according to a modified protocol

(40). These cells were then maintained in hybridoma medium (IMDM medium with

10% fetal calf serum, 5 mglL bovine insulin, 10 mg/L bovine transferrin, 17.3 I-lg/L

sodium selenite, and 5.5 x10-5M 2ME) for up to 30 days. Plasma cell morphology

was confinned by cytospin (differential staining and intracellular immunoglobulin),

ELISPOT and flow cytometry (both described below). Plasma cells at different stages

of differentiation were harvested for further study.

3H-Leucine (H-Leu) incorporation assay to measure total protein synthesis after IT

treatment

A total of 105 cells/well were incubated with ocCD22-IT, ocCD38-IT, or ocCD25-IT

(as a control) at concentrations from 10-13M to 1O-8M for 1 hour at 4°C. Cells were

washed and cultured overnight in RPMI with 10% fetal calf serum for B cells or with

addition of 2.5 ng/ml IL-2 and 100 ng/ml IL-IO for plasma cells at 37'C with 5%

C02. Cells were washed again, and pulsed in Leucine-free RPMI with 5 fICi/well 3H_

Leu for 5hr (for Daudi cells) or 18 hours (for B and plasma cells) at 37'C. 'H-Leu

incorporation was examined by harvesting cells on to filters and counting the

radioactivity by beta-counter (Wallac, Gathersburg, MD).

ELISPOT for detection of onti-aGol Ab and total immunoglobulin production

For detection of anti-aGal Ab production, 96-well MultiScreen-HA plates (MAHAS

4510 mixed cellulose esters, Millipore, Bedford, MA) were coated with 100 fII/well (5

fIg/ml in PBS) of aGal-BSA or control BSA (Alberta Research Council) at 4'C

91

Section ll/- Chapter 4

overnight. For detection of total IgM or IgG production, goat anti-human IgM or IgG

(Southern Biotech, Birmingham, AL) were used as coating reagents with goat anti

mouse IgM or IgG as negative controls. Plates were washed with PBS and blocked

with IMDM and 0.4% BSA for I hr at 37'C. A total of Ixl06 cells/well, with a serial

5-fold dilution, were cultured overnight in a modified hybridoma medium (10% fetal

calf serum replaced by 0.4% BSA) at 37°C with 5% CO,. The plates were then

washed with PBS and PBS plus 0.1 % Tween-20. Antibody production was detected

with 100 J.tl goat anti-human IgM or IgG conjugated to HRP (Southern Biotech) at

1:1000 dilution in PBS with 1% BSA and 0.5% Tween at 4'C for I hour. Plates were

washed with PBS plus 0.1 % Tween and PBS. Spots were visualized with substrate

ABC or 4CN (Sigma) under a stereomicroscope (Nikon SM2-U) equipped with a

vertical white light. Data were presented as spot forming units (SFU) per 106 cells.

Flow cytometry

Flow cytometry to detect B and T cells was performed on blood, BM (aspirates

obtained from the iliac crests), and LN (biopsies obtained from either inguinal or

axillary regions). The direct conjugated antibodies anti-CD3 FITC (FNIS, kindly

provided by Dr. David Neville, Bethesda, MD) and anti-CD2 PE (Leu-5b, Becton

Dickinson) were used as T cell markers, and anti-CD20 FITC (Leu-I 6, Becton

Dickinson) and anti-CD22 PE (Clone RFB4, Caltag Laboratories, Burlingame, CAl as

B cell markers. Blood and BM were incubated at 4° C, lysed at room temperature with

ACK-Iysing buffer (Bio-Whittaker, Walkersville, MD), and washed and resuspended

in 500 J.lI FACS Medium (1% BSA and 0.1% azide in phosphate-buffered saline).

LNs were mashed, filtered, and resuspended in 500 J.lI FACS medium. Cell count was

approximately 1 x 106 / 100 III in all samples. The samples were stained using the

aforementioned T and B cell-specific antibodies. Acquisition was perfonned under hi

flow using the F ACScan (Becton Dickinson), and samples were analyzed using

WinList (Verity Software House, Inc., Topsham, ME).

92


Cell depletion using magnetic beads

One million cells (106/ml) were incubated with 10 fig mAb (targeting the cells that

were to be depleted) at 4°C for 1 hour. Cells were then washed and incubated with

secondary goat-anti-mouse IgG magnetic beads following the manufacturer's protocol

(Dynal). The cells that bound to the mAb were removed, and the remaining cells were

assessed by flow cytometry and for antibody production by ELISPOT.

Measurement of anti-aGal antibody (anti-aGal Ab) by ELISA

Daily serum samples were obtained and anti-a Gal Ab levels were measured by

ELISA (19). This consisted of loading a 0.016%-2% concentration of baboon serum

on Maxisorb plates (Nunc, Naperville, IL, USA) coated with 5 flg/mL of aGal-BSA

(Alberta Research Council). Bound antibodies were detected using polyclonal donkey

anti-human IgG (Accurate, Westbury, NY) or rabbit anti-human IgM (DAKO,

Copenhagen, Denmark) conjugated to horseradish peroxidase. Color development

was achieved by using o-phenylenediamine dihydrochloride (Sigma) as a substrate at

0.9mg/mL in PBS with urea hydrogen peroxide (Sigma). Absorbance at 490 run was

determined by a THERMOmax plate reader (Molecular Devices, Menlo Park, CAl

after blocking the reaction with 2N H2S04.

Immunohistochemistry

For in vitro experiments, a total of 100,000 cells in 200 fll PBS was centrifuged onto

slides by Cytospin. The slides were either immediately stained with Giemsa or fixed

overnight in 0.25% paraformaldehyde at 4° C for intracellular immunoglobulin

staining. Goat anti-human IgM and/or IgG conjugated to peroxidase (Southern

Biotech) at 1: 1000 was added to the slides after which they were incubated at room

temperature for 1 hour in a humidified chamber. After slides were washed, a DAB

substrate kit (Vector Laboratories, Burlingame, CA) was used to visualize the

staining.

For in vivo experiments, depletion of CD20 positive cells in LNs was assessed using

the anti-CD22 anti-human mAb (Caltag) as primary antibody on frozen samples in a

standard immunohistochemical assay. Depletion of CD22-positive cells was

93

Section IIJ- Chapter 4

examined in paraffin-embedded tissue using the anti-CD20 anti-human mAb (DAKO,

Carpinteria, CAl.

RESULTS

In vivo experiments

Effect oJWBI on B cell depletion and anti-a(}al Ab production

Administration of 2xl50 cGy WEI led to rapid and complete B cell depletion as

detected by flow cytometric analysis of blood and BM by day 2, with recovery

starting by day 28 in the blood and BM (data not shown). B cell depletion in the LN

was 75 - 100% between days 7 and 14, with recovery by day IS (data not shown).

This finding was confinned by immunohistochemistry, where complete depletion of

B cells in the LN was observed at day 9, with recovery starting at day IS in the

periphery of the follicles (See Chapter 3, Figure 2). In these baboons, a

thrombocytopenia «20,000/f!1) was noted between days 6 and 13, and a

leukocytopenia « 1000/f!1) between days 13 and 17, most probably due to BM

depression. The anti-aGal Ab levels in these baboons remained unchanged, in nonnal

ranges (data not shown), although no EIAs were performed. When EIAs were

performed after myeloablative WEI (900 cGy), the rate of recovery of anti-aGal Ab

was unchanged when compared to EIA treatment alone (data not shown). These data

confirm that B cells are readily depleted by WEI in blood and BM for 28 days, and in

LN for 15 days, but that this depletion is not associated with a significant decrease in

anti-a Gal Ab production.

Effect oJthe a:CD22-IT on B cell depletion and anti-a(}al Ab production

At the doses given, ocCD22-IT was efficient in depleting B cells in blood and BM

(86-99% (Fig. 1 - top) and 80-97%, respectively) between days 6 and 8. B cell

depletion in LN was less marked, the maximum percentage of depletion ranging

between 50 and 58% on days 7-12 (data not shown). T cell counts in blood, BM, and

LN remained unchanged. However, the recovery of B cells was swift. In blood,

evidence of recovery was observed at.day 12 in both baboons, with increases up to 6-

94


fold baseline levels between days 62 and 85 (Fig I - top). In BM, recovery was first

observed between days 12 - 15, with recovery to baseline levels by day 62 (Data not

shown).

Figure 1.

150

1 120

90 60 25

-4 o

fu EIA

2 6 8

",COOl·IT

15

__ lgM

___ lgG

25

15 22 28 36 62 85

Days

35 45 55 65 75 85

Days

The effect of ccCD22-IT in vivo on B cell depletion and anti-aGal Ab production in a baboon.

Top. Bar chart demonstrating the depletion of CD20-positive B cells in blood (as absolute cell count)

before, during, and after treatment with four iv doses of 0.19 mg/kg every other day of ccCD22-IT

commencing on day O. Complete depletion ofB cells was seen immediately following treatment at day

Bottom. Anti-aGal Ab profile of this baboon. EIAs were performed at time of maximum B cell

depletion (days 8, 9, and 10). Note the complete depletion of anti-aGal IgG and IgM Ab following

each consecutive EIA. The maximum depletion was 40% and 45% 7 days following ElA for IgG and

IgM, respectively. Rebound of anti-aGal IgG and IgM Ab to levels 12- and 9-fold higher than baseline,

respectively, was observed by day 83.

95


A rebound of B cells to numbers higher than baseline was also observed in LN in one

baboon by day 22 (2-fold), while in a second baboon return to baseline levels was

seen after 18 days (data not shown). Immunohistochemistry of LN correlated well

with the flow cytometry data (data not shown). In one baboon, the initial anti-aGal

Ab profiles were encouraging with depletion of IgG and IgM 48 hours post EIA

sustained at 70 and 100%, respectively. However, by day 7, the depletion of IgG and

IgM was 17 and 34%, respectively (data not shown). In the second baboon, the

maximum depletion of anti-aGal Ab was 40 - 45% at 7 days, with rebound above

baseline levels by day 55 (Fig 1 - bottom). Both baboons developed anti-murine

antibodies and one baboon developed anti-ricin A chain antibodies, as determined by

ELISA (data not shown). No changes in platelet or white cell count were observed.

These data indicate that although the ocCD22-IT was effective in the short-term

depletion of B cells from blood, BM, and LN, this was not associated with a

significant decrease in the rate of return of anti-aGal Ab. Furthermore, treatment with

this IT led to the development of anti-murine and anti-ricin antibodies.

Effect of the cx:CD38 mAb on B cell depletion and anti-aGal Ab production

Cell coating with the ocCD38 mAb was 100% throughout the treatment in the one

baboon studied. However, no substantial changes occurred in anti-aGal Ab levels

following EIA (data not shown). Although blood levels of anti-CD38 mAb

approximated 1000 ng/ml, no sensitization to the murine component of the

monoclonal antibody occurred. Conjugation to the ricin A toxin would, therefore,

seem necessary to successfully deplete B cells and plasma cells in vivo.

Effect of the cx:CD38-IT on B cell depletion and anti-aGal Ab production

ocCD38-IT was first tried in a baboon which, 18 months before, had received

myeloablative \VBI and had undergone treatment with melphalan for several weeks.

However, by the time of treatment with the ocCD38-IT, anti-aGal Ab levels, as well

as T and B cell counts, had returned to baseline levels (i.e., pre WEI). Following

96


depletion by EIA, anti-aGal IgG and IgM Ab remained at undetectable levels for I

week. After an additional EIA on day 26, anti-aGal IgM Ab was undetectable for 10

days while the IgG returned to baseline values by day 4 (Fig. 2). In a second,

previously untreated baboon, anti-aGal IgO Ab increased to levels greater than

baseline within 48 hours after the last of 3 consecutive EIAs (data not shoVvn).

Coating ofCD38-positive cells with ocCD38-IT was 100% in both cases. Sensitization

occurred in both baboons, with the development of anti-murine and anti-ricin

antibodies. Treatment with the ocCD38-IT in a third baboon did not result in coating

of CD38-positive cells and sUbtherapeutic plasma levels of ocCD38 were measured

(data not shovm). The reasons for failure in this animal were unclear.

12

9 __ lgG

"iii~ ---a--lgM

(,!)-ij E ,- s .- en

..... :::t

~-3

o~--~--~~--~--~~--~---c -5 5 15 25 35 45 55 m i Days

EIA EIA

Figure 2.

Anti-aGal Ab profile in baboon receiving ocCD38-IT. During iv treatment with ocCD38-IT (0. J

mg/kg/day for 14 days commencing on day 0), 3 consecutive EIA were performed, effectively

removing all anti-aGal IgG and IgM Ab from circulation. Levels of anti-a Gal Ab remained

undetectable for 7 days after which recovery of both IgO and IgM occurred, with rebound above

baseline of IgG. After a fourth EIA was performed on day 26. IgM was again undetectable for 10 days.

whereas IgO returned to previous levels after 6 days.

97

Section 111- Chapler 4

These results indicate that in one experiment ocCD38-IT may have been effective in

depleting antibody-producing cells, as the levels of anti-aGal Ab remained at low

levels following EIA. The reason for our failure to duplicate these results in

subsequent baboons are unclear. It is possible that the development of anti-murine

and/or anti-ricin A chain antibodies played a role. Alternatively, it is possible that the

positive effect observed with ocCD38-IT in one baboon may have been associated

with previous treatment (which includes melphalan), even though the drug has been

discontinued> 2 months previously.

Effect of ocCD20 mAb and/or WBI on B cell depletion and anti-aGal Ab production

Following the first dose of ocCD20 mAb, B cells were rapidly depleted from the blood

in all baboons, and remained at low levels through day 112 (Fig. 3 - top). Depletion of

B cells in BM increased following each dose of ocCD20 mAb, with a maximum

depletion of 92% at day 40 in one baboon, after which recovery was observed (data

not shown). The effect of ocCD20 mAb on B cell depletion in LN was less impressive,

with a maximum depletion of 70% B cells/gram LN at day 35, and recovery to

baseline values by day 57. In one baboon, because some return of B cells in BM was

observed at day 35, 150 cGy WEI was administered at day 42. Complete depletion of

B cells in BM was achieved until day 105, with 40% recovery at day 287. The

depletion in LN was complete until day 70, with recovery by day 105 (data not

shown). Although EIA resulted in complete depletion of anti-aGal IgG and IgM Ab,

recovery of anti-a Gal Ab could be observed within 72 hours following the last EIA

(Fig. 3 - bottom). Three days following EIA, the reduction in anti-aGal IgG Ab levels

was 80%, with recovery to 75% of pre-treatment levels within 2 days thereafter. The

reduction in anti-aGal IgM Ab levels was less marked (60% 3 days following EIA),

with recovery to pre-treatment levels within 1 day, and increase above baseline levels

(1-2 fold) within 5 days.

98


In vitro experiments

Morphology of B and plasma cells in culture

The anti-human ocCD20 mAb, ocCD22 mAb, surface immunoglobulin, and ocCD38

mAb exhibited good cross-reactivity in binding to baboon spleen B cells when

compared with binding to human spleen B cells (data not shown). During plasma cell

differentiation, C020, C022, and surface immunoglobulin were down~modulated

whereas CD38 expression remained high (Fig. SA). Morphologically, plasma cells

could readily be distinguished from B cells by size and cell-cell interactions (Fig. SB).

Furthermore, plasma cells secreted immunoglobulin, as was confirmed by

intracellular immunoglobulin staining (data not shown).

These results indicate that the anti-human ITs and rnAb used in these studies cross

react with baboon cells, and that efficient CD38 targeting may be effective in

depleting both B and plasma cells.

Inhibition of protein synthesis

The ability of ocCD22-IT and ocCD38-IT, along with the negative control ricin A

conjugated anti-CD2S mAb (ocCD2S-IT), to inhibit protein synthesis using the human

Burkitt's lymphoma, Daudi, cell line was evaluated. Flow cytometric analysis of

Daudi cells demonstrated that they are positive for both CD22 (RFB4, 66%) and

CD38 (OKTlO, 74%), and that CD38 stains brighter than CD22 (data not shown).

Using the 3H-Leucine incorporation assay, 100% inhibition of protein synthesis was

achieved with ocCD22-IT at 10,9 - 10'8 M, whereas ocCD38-IT led to a maximum of

60% inhibition of protein synthesis at 10'8 M (Fig. 4). Furthermore, the slope of the

curve for ocCD38-IT is much less steep than that for ocCD22-IT. This suggests that

ocCD38-IT has either a slow rate of internalization and intracellular routing, or slow

kinetics, leading to less effective inhibition of protein synthesis.

99

Section II! - Chapter 4

• . ~ :t= ::::-

~ '" 8.:» No. N-c-O § • 0 _ u

.2= WBl150 cGy o • ~ u

! .c .. ·1 2 7 14 21 42 57 71 10S 14S 216 287

G • Days ",CD20 mAb

"1 ___ lgG

" __ lgM

40

" " C!l

~ 30 1 ~

"" c -'" " « " "

0 0 " 40 " " , 00 '" nt t Days

EIA

Figure 3.

The effect of <xCD20 mAb in vivo on B cell depletion and anti-aGal Ab production in a baboon.

Top. Bar chart demonstrating the depletion of CD22-positive B cells in blood (as absolute cel! count)

before, during, and after treatment with four iv doses of20 mg/kg of <xCD20 mAb, at weekly intervals

commencing on day O. Almost total depletion of B cells was seen immediately after initiating the

course of <xCD20 mAb therapy. As some return of B cells was seen in the BM by day 35, WBI (150

cGy) was administered - this led to almost complete depletion of B cells> 3 months.

Bottom. Anti-aGal Ab profile in this baboon. EIAs were perfonned at time of maximum B cell

depletion. Note the complete depletion of anti-aGal Ab following each consecutive ElA. Return of

anti-a.Gal Ab was not significantly inhibited following treatment with <xCD20 mAb, with anti-aGal

IgG Ab returning to baseline values, and anti-aGal IgM Ab increasing to levels above baseline. The

maximum reduction in anti-aGal IgG Ab was 80% 72 hours following EIA, with recovery to 75% of

pre-treatment levels within 2 days. The maximum reduction in anti-ctGal IgM Ab was less marked,

reaching 60% 72 hours following EIA, with recovery to pre-treatment levels within 1 day, and increase

above baseline levels (1-2 fold) within 5 days.

100


Activated B cells andlor early plasma cells had high levels of protein synthesis (in

contrast to resting B cells). Protein synthesis was efficiently inhibited by the ocCD22-

IT by approximately 60% at 10-10 M in suspensions containing 80% CD22-positive

cells (Fig. 5). This inhibition was less marked than that observed with the ocCD22-IT

on human Daudi cells, presumably due to the presence of a CD22-negative population

in the baboon cell preparation. Plasma cells down-modulated surface CD22

expression during differentiation. Consequently, at day 8 (22% CD22-positive cells)

and day 15 (6% CD22-positive cells) of in vitro differentiation, protein synthesis was

not inhibited by the ocCD22-IT.

These data suggest that ocCD22-IT is more effective than ocCD38-IT in inhibiting

protein (e.g., antibody) synthesis in vitro, but, as predicted, that it is not effective in

inhibiting protein synthesis of differentiated B cells (e.g., plasma cells).

Anli-aGol Ab production by baboon spleen cells after depleting CD20-, CD22-, or

CD38-positive cells

Baboon spleen cells, depleted of T cells, monocytes, and granulocytes, were used in

this assay. Removal of CD20-positive B cells from baboon spleen cells with magnetic

beads resulted in an increased frequency of anti-aGal IgM Ab and total

immunoglobulin secretors (Fig. 6A). This effect was also seen when CD22-positive

cells were depleted (data not shown). Depletion ofCD38 cells by magnetic beads led

to a significant decreased frequency of anti-a Gal Ab and total immunoglobulin

secretors (Fig. 6B). Flow cytometry confirmed the efficient removal ofCD20-, CD22-

,or CD38-positive cells (data not shown).

101


100

75

50

25 : 0+--,--,--,---.--,--,--14 -13 -12 -11 -10 -9 -8 -7

mAb concentration (2 x 10" M)

Figure 4.

--'>- oc CD22-IT

--D- oc CD38-IT

--- oc CD25-IT

Protein synthesis by human Daudi cells in the presence of ocCD22-IT, :x:C038-IT, and the negative

control :x:CD25-IT. Data are presented as percentage protein synthesis ( i.e., eH-Leu incorporation by

cells treated with IT j 3H_Leu incorporation by cells treated with unconjugated mAb) x 100%).

The negative control ocCD25-IT demonstrates a maximum and stable 75% protein synthesis at all

concentrations of antibody. Treatment with ocCD22-IT leads to an efficient decrease in protein

synthesis, approaching 0% at concentrations between IO-lO_IO-sM, whereas treatment with ocCD38-IT

is not as efficient. The minimum protein synthesis is 40% at a concentration of ! O·sM.

These data indicate that enrichment for plasma cells (by depleting CD20- or CD22-

positive cells) leads to a relative increase in the frequency of anti-aGal Ab and total

immunoglobulin producing cells, aud that depletion of CD38-positive cells (i.e., B

cell blasts and plasma cells) removes auti-aGal Ab aud total immunoglobulin

secretors. Plasma cells and B cell blasts therefore appear to be responsible for the

majority of anti-aGal Ab production in baboon spleens.

102

c: 'iii -0 ~

a. In

'0 'iii "

100

75


---- Day 3 (80% CD22+)

~ Day 8 (22% CD22+)

- Day 15 (6% CD22+)

c:.t: 0- 50 ,_ c: ~>o ,Coo :;: .5 ~ •

25

0+-~~-4L-~~~-,--,-, -16 -15 -14 -13 -12 -11 -10 -9 -8 -7

mAb concentration (2 x 10" M)

Figure 5.

Protein synthesis by baboon spleen cells in the presence of ocCD22, ocCD22-IT, on days 3, 8, and 15

after in vitro activation and differentiation as measured by 3H_Leu incorporation. Data are presented as

percentage protein synthesis ( i.e., eH-Leu incorporation by cells treated with ocCD22-IT ! 3H-Leu

incorporation by cells treated with xCD22) x 100%). The minimum percentage protein synthesis was

achieved at day 3 after in vitro differentiation, correlating with 80% CD22-positive cells. At days 8 and

15 after in vitro differentiation, protein synthesis was not significantly changed. At these timepoints,

only 22% and 6% CD22-positive cells, respectively, were measured.

DISCUSSION

It is well recognized that anti-aGal Ab are the major barriers to clinical

implementation of xenotransplantation. Anti-aGal Ab are believed to be responsible

for both hyperacute and acute vascular rejection of vascularized, discordant

xenografts (9,14), Although it is possible to remove anti-aGal Ab from the circulation

of nonhuman primates, either by perfusing the recipient's blood through porcine

organs or through an extracorporeal immunoaffinity column specific for anti-aGal

Ab, sustained depletion of anti-a Gal Ab has not been achieved due to ongoing

antibody production (6). Treatment modalities aimed at reducing the production of

anti-aGal Ab have to date been unsuccessful. Various pharmacologic agents,

including brequinar sodium, methotrexate, melphalan, cladribine, zidovudine,

cyclophosphamide, and mycophenolate mofetil, have been evaluated in our laboratory

103

Section flI - Chapter 4

without clinically significant reduction of antibody production (6,21). In our pig-to

primate model aimed at inducing immunological tolerance, we have recently

demonstrated that the anti-CDl54 mAb effectively prevents the induced (T cell

dependent) antibody response to porcine antigens, but the return to baseline levels of

(T cell-independent) anti-aGal Ab, in particular anti-aGal IgM Ab, is not prevented

(6,20). Following depletion, the return of anti-aGal Ab in vascularized organ

transplant models is invariably associated with destruction of the transplanted graft

(18,19,41).

Our center has been interested in studying the cellular origin of anti-aGal Ab. We

have examined numerous tissues obtained from naIve baboons and baboons

previously exposed to porcine tissue to determine whether anti-aGal Ab-producing

cells are specific for, or restricted to, certain anatomical regions of the body.

ELISPOT analyses of these tissues were performed and we observed the highest

frequencies of anti-aGal Ab (predominantly IgM) secretors in naIve baboons in the

spleen, tonsils, BM, and LN. Anti-aGal IgG was primarily found in the LN, whereas

anti-aGal IgA was mostly seen in the small intestine in Peyer's patches. In

splenectomized baboons sensitized to porcine antigens, however, these secretors

(especially IgM and IgG) were primarily observed in the BM (Xu Y et aI.,

unpublished data). We believe that, although treatment aimed specifically at cells

producing anti-a Gal Ab is highly desirable, the technology to do this is not yet

available. Attention was, therefore, directed at less specific therapies targeting all

antibody-producing cells.

104

<: o

7.5


~-0'"

--<>- medium

Co 0 5.0 ~ ~ -'1- oc CD22 o ><

" .5 E Co

-ocCD22-IT

~ .!!.. 2.5 ...J , J:

'"

::J LL rJ)

0.0 +--.-----r-,---,----,---,,--,---.-----, -16 -15 -14 -13 -12 -11 -10 -9 -8 -7

mAb concentration (2 x 10n M)

Post

" " 0 0

'" ~ Q)

c. c. Q) Q) "C "C ., ., '" '"' 0 0 u u " -

Figure 6

_ anti-a GaligM

_totallgM

=totallgG

ELiSPOT assay demonstrating the number of spot fanning units (SFU) / 106 cells of anti-aGal IgM

Ab, and total 19O and 19M in baboon spleens after depletion of CD20- or CD38-positive cells by

magnetic beads.

A. Depletion of C020-positive cells resulted in an approximate 30-fold increase of anti-aGal IgM Ab

production over baseline, and a 5-9 fold increase of total IgG and IgM. This increase indicated that the

non-depleted (C020-negative) cells were responsible for the majority of antibody production.

B. Depletion of CD38-positive cells led to an 88% decrease of anti-aGal IgM Ab, and a 96-98%

decrease of total IgG and IgM production, respectively. These data suggest that CD38-positive cells are

mainly responsible for antibody production.

105


Recently, significant progress has been made in the treatment of several hematologic

malignancies. Several in vitro and in vivo pre-clinical and clinical studies have shovm

promising data using specific anti-B and/or plasma cell mAbs to reduce the tumor

load of B cell malignancies (22,23,24,25,42), We, therefore, assessed the capacity of

several of these mAbs and ITs to deplete nonnal (non-malignant) B and/or plasma

cells and to reduce production of anti-aGal Ab in baboons.

Both treatment with the ocCD22-IT in vivo and \VBl led to a complete, but transient,

depletion of CD22- and CD20-positive cells in blood and BM, Furthennore, there was

no substantially reduced anti-aGal Ab production following EIA. In fact, after the

ocCD22-IT treatment, as ocmurine antibodies developed, the levels of anti-aGal Ab

increased significantly above baseline. It is not clear if this was due to sensitization to

murine-a Gal, or a rebound phenomenon after depletion of B cells. The latter seems

more likely as the amount of total inununoglobulin also increased.

Sustained B cell depletion was achieved when ocCD20 mAb was combined with 150

cOy \VEL B cells were virtually undetectable in the circulation for> 3 months, and

were almost completely depleted from BM and LN for at least 70 days. Recovery in

the blood and BM had not yet reached 25 and 40 % baseline, respectively, at day 216.

Despite this medium-term, near-complete depletion of B cells, reduction of anti-aOal

Ab levels was not sustained. The maximum depletion of anti-aGal IgG and IgM Ab

following 72 hours following EIA was 80% and 60%, respectively, and was sustained

for only 2 days, after which there was a return to baseline levels of IgM. Levels of

IgO, however, remained at 66% of those pretreatment. Since depletion of B cells with

the ocCD22-IT was not as effective as that obtained with the ocCD20 mAb, we did not

combine the ocCD22-IT with WEI.

These findings suggest that CD20- or CD22-positive B cells are not the major source

of anti-aGal Ab production, and that other cells, presumably plasma cells or B cell

blasts, are mainly responsible for antibody production. These observations are also in

keeping with data presented by other groups, suggesting that antibody-producing cells

106


are long-lived (43,44) and radiation-resistant (45). Unfortunately. in vivo treatment of

baboons with ocCD38-IT, reportedly targeting plasma cells, did not yield reproducible

results. Only in one of 3 baboons was treatment with ocCD38-IT followed by reduced

production of anti-a Gal Ab. It is lU1clear, however, whether this reduction in

production of anti-aGal Ab was due solely to the treatment with cx:CD38-IT, or to

some extent due to a late effect of extensive prior inunlU10suppressive treatment. In

two other cases, when naiVe baboons were used, no effect on antibody production was

observed. One potential explanation is that the ocCD38 mAb interaction with the

CD38 molecule expressed on the cell surface did not efficiently mediate

internalization of the mAb - receptor complex, which is required for effective killing

by dgR T A. Furthermore, as CD38 is also expressed on cells other than B or plasma

cells (e.g., activated T cells, natural killer cells, and BM cells), this could interfere

with the specific binding of ocCD38-IT to B cells and plasma cells, reducing its

efficacy. However, this explanation is not likely, since all cells were coated with

ocCD38. In these experiments, higher doses of ocCD38-IT may have been more

effective by providing greater cross-linking of receptors which could lead to increased

internalization of the IT.

The results of these in vivo studies prompted us to carry out further in vitro

investigations. We first demonstrated that expression of CD20, CD22, and CD38 by

baboon B cells is highly dependent on the state of differentiation I maturation of the

cells. Most B cells express immunoglobulin, CD20, CD22, and CD38 on their

surface. Plasma cells, however, have down-modulated surface immunoglobulin,

CD20, and CD22 expression, although expression of CD38 is maintained on baboon

plasma cells. This suggests that mAbs or ITs directed against CD20 or CD22 will

potentially deplete B cells but, if both B- and plasma cells are to be depleted, a mAb

or IT specific for a plasma cell marker, such as CD38, would be necessary.

We confinned this in our subsequent experiments. Removal of CD38-positive cells

from baboon spleen B lineage cells by magnetic beads led to a greatly diminished

frequency of anti-aGal IgM Ab and total immunoglobulin secretors. In contrast,

107

Section IIl- Chapter 4

depletion of CD20- or CD22-positive cells from baboon spleen B lineage cells led to

an effective enricbment of CD20- or CD22-negative cells and to an increased

frequency of anti-aGal IgM Ab and total immunoglobulin secretors. 'When the

inbibition of protein synthesis by human Daudi cells with ocCD38-IT was compared

to the inbibition obtained with ocCD22-IT, however, we found that ocCD38-IT was

less effective than ocCD22-IT. These data may correlate with our inconsistent in vivo

experience with ocCD38-IT. It appears that, although ocCD38-IT targets the desired

population of (plasma) cells, it is not particularly efficient at killing those cells,

possibly due to low affinity interaction of ocCD38 mAb or slower internalization of

the CD38 receptor. Unfortunately, we have not been able to find other anti-human

anti-CD38 rnAb that cross-react with baboons. In contrast, ocCD22-IT is very

effective at killing CD22-positive cells but, unfortunately, these cells are not the

major secretors of antibody, including anti-aGal Ab.

In none of the in vivo experiments described above, where administration of anti-B

cell agents combined with EIA +/- \VBI was the only therapy, were any infections

noted. In one case (not included in this study), treatment with xCD20 mAb was

incorporated in our protocol aimed at inducing immunological tolerance (20,41,46).

This protocol consists of induction therapy with splenectomy, \VBI 300 cGy, ATG,

and EIAs, and maintenance therapy with anti-CDl54 mAb +1- CyA, mycophenolate

mofetil, corticosteroids, and cobra venom factor. Although this is an intensive

protocol that renders the baboon severely immunosuppressed, viral infections have

rarely been observed. However, when pretreatment with ocCD20 rnAb was added, a

viral hepatitis (confirmed by inclusion bodies on histology) resulted in death of the

baboon within 20 days. It is conceivable that pretreatment with ocCD20 mAb reduced

the ability of the baboon to mount a humoral response to the viral antigen load. Close

monitoring would, therefore, be necessary if ocCD20 rnAb therapy were to be

combined with other intensive immunosuppressive therapies.

More recently, several groups have demonstrated that combining anti-B ITs, e.g., anti

CD19 and anti-CD22 (47), or combining anti-B cell and anti-plasma cell ITs, e.g.,

108


anti-CD19, anti-CD22, and anti-CD38 (48), can cure B cell neoplasias in a mouse

tumor xenograft model, while the individual ITs are unable to do so alone. This

treatment could overcome the heterogeneity of surface antigen expression on B cells

and plasma cells and prove valuable in our goals to deplete antibody-producing cells.

An interesting observation was also recently made by Treon et al (49). They found

that treatment with interferon-gamma caused significant upregulation of CD20-

expression on plasma cells of patients with mUltiple myeloma. This approach (i.e.,

pretreatment with interferon-gamma) could, therefore, render some plasma cells more

susceptible to treatment with ocCD20 rnAb. In vitro studies at our center, however,

have so far failed to demonstrate significant upregulation of CD20 on baboon plasma

cells by interferon-gamma therapy (Thall A, unpublished data).

In conclusion, the above data demonstrate that baboon B cells can be efficiently

depleted with WBI, ocCD22-IT, or ocCD20 mAb, and that medium-term (>3 months)

depletion of B cells can be achieved with ocCD20 mAb combined with WBI.

However, depletion ofB cells does not lead to a clinically significant reduction in the

rate or extent of return of anti-a Gal Ab following EIA. These observations suggest

that B cells are not the major source of anti-aGal Ab production, and that attention

must be directed towards suppressing plasma cell and B cell blast function. We have

demonstrated in vitro that CD38-positive cells are the major secretors of anti-aGal

Ab. Treatment with ocCD38-IT, however, did not yield consistent results in vivo,

possibly due to pre-existing anti-ricin rnAbs, low-affinity or slow internalization of

the antibody, sub-optimal conjugation of ricin-A, and/or relatively low specificity of

CD38. Nevertheless, further studies focussed on this IT or on the development of new

and refined anti-plasma cell agents are clearly warranted and are currently in progress

at our center. Furthennore, combinations of ocCD20 rnAb or ocCD22-IT with an

optimized anti-plasma cell IT may be of benefit.

109


REFERENCES

1 Cooper DKC. Xenografting; how great is the clinical need? Xeno 1993 ; I; 25 2 Cooper DKC, Ye Y, Rolf LL Jr, et al. The pig as a potential organ donor for man. In: Cooper DKC,

Kemp E, Reemtsma K, et al. (eds.). Xenotranplantation. Heidelberg, Springer 1991. p481 3 Sachs DH. The pig as a potential xenograft donor. Vet Imm Immunopath 1994 ;43: 185 4 Platt lL, Fischel RJ, Matas Al, et al. Immunopathology of hyperacute xenograft rejection in a swine

to-primate model. Transplantation 1991; 52: 514 5 Bach FH, Robson SC, Winkler H, et al. Barriers to xenotransplantation. Nat Med 1995; 1: 869 6 Alwayn IPl, Basker M, Buhler L, Cooper DKC. The problem of anti-pig antibodies in pig-to-primate

xenografting: current and novel methods of depletion and/or suppression of production of anti-pig antibodies. Xenotransplantation 1999; 6: 157

7 Schmoeckel M, Bhatti FN, Zaidi A, et al. Orthotopic heart transplantation in a transgenic pig-toprimate model. Transplantation 1998; 65: 1570

8 Zaidi A, Schmoeckel M, Bhatti F, et al. Life-supporting pig-to-primate renal xenotransplantation using genetically modified donors. Transplantation 1998; 65: 1584

9 Good AH, Cooper DKC, Malcolm Al, et al. Identification of carbohydrate structures that bind human anti-porcine antibodies: implications for discordant xenografting in humans. Transplant Proc 1992; 24: 559

10 Oriol R, Ye Y, Koren E, et al. Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation. Transplantation 1993; 56: 1433

II Galili U. Interaction of the natural anti-Gal antibody with agalactosyl epitopes: a major obstacle for xenotransplantation in humans. Immunol Today 1993; 14: 480

12 Galili U, Clark MR, Shohet S8, et al. Evolutionary relationship between the natural anti-Gal antibody and the Galal--,;>3Gal epitope in primates. Proc. Natl. Acad. Sci. USA 1987; 84: 1369

13 Rose AG, Cooper DK. A histopathologic grading system of hyperacute (humoral, antibodymediated) cardiac xenograft and allograft rejection. 1 Heart Lung Transplant 1996; 15: 804

14 Galili U. Anti-agalactosyl (anti-Gal) antibody damage beyond hyperacute rejection. In: Cooper DKC, Kemp E, Platt JL, et al. (eds). Xenotransplantation, 2nd edition. Heidelberg, Springer 1997. p 95

15 Taniguchi S, Neethling FA, Korchagina EY, et al. In vivo immunoadsorption of anti pig antibodies in baboons using a specific Galal-3Gal column. Transplantation 1996; 10: ]379

16 Kozlowski T, lerino FL, Lambrigts D, et al. Depletion ofanti-aGall-3Gal antibody in baboons by specific immunoaffinity columns. Xenotransplantation 1998; 5: 122

17 Sablinski T, Latinne D, Giane!1o P, et al. Xenotransplantation of pig kidneys to nonhuman primates: I. Development of the model. Xenotransplantation. 1995; 2: 264

18 Cooper DKC, Cairns TDH, Taube DH. Extracorporeal immunoadsorption of anti-pig antibody in pigs using aGal oligosaccharide immunoaffinity columns. Xeno 1996; 4: 27

19 Xu Y, LorfT, Sablinski T, et al. Removal of anti-porcine natural antibodies from human and nonhuman primate plasma in vitro and in vivo by a Gala 1-3Gal~ 1-4Glc-X immunoaffinity column.

Transplantation 1998; 65: 172 20 Buhler L, Awwad M, Basker M, et a1. High-dose porcine hematopoietic cell transplantation combined

with CD40L blockade in baboons prevents an induced anti-pig humoral response. Transplantation 2000. In press

21 Lambrigts D, Van Calster P, Xu Y, et al. Pharmacologic immunosuppressive therapy and extracorporeal immunoadsorption in the suppression of anti-aGal antibody in the baboon. Xenotransplantation 1998; 5: 274

22 Vitetta ES, Fulton RJ, May RD, et al. Redesigning nature's poisons to create anti-tumor reagents. Science 1987: 238: 1098

23 Ghetie MA, Tucker K, Richardson J, et a1. The antitimor activity of an anti-CD22 immunotoxin in SCID mice with disseminated Daudi lymphoma is enhanced by either an anti-CD 19 antibody or an anti-CD 19 immunotoxin. Blood 1992; 80: 2315

24 Grossbard ML, Press OW, Appelbaum FR, et al. Monoclonal antibody-based therapies of leukemia and lymphoma. Blood 1992; 80: 863

25 Pastan I and Fitzgerald D. Recombinant toxins for cancer treatment. Science 1991; 254: 1173

110


26 Dewey WC, Ling CC, Meyn RE. Radiation-induced apoptosis: Relevance to radiotherapy. Int J Rad Oncol BioI Phys 1995; 33: 781

27 Thorpe PE, Wallace PM, Knowles PP, et al. Improved antitumor effects of immunotoxins prepared with deglycosylated ricin A-chain and hindered disulfide linkages. Cancer Res 1988; 48: 6396

28 Sausville EA, Headlee D, Stetler-Stevenson M, et al. Continuous infusion of the anti-CD22 immunotoxin IgG-RFB4-SMPT-dgA in patients with B-celllymphoma: a phase I study. Blood 1995;85:3457

29 Campana D, Janossy G, Bofill M, et at. Human B cell development. L Phenotypic differences of B lymphocytes in the bone marrow and peripheral lymphoid tissue. J Immunol 1985; 134: 1524

30 Malavasi F, Funaro A, Roggero S, et at Human CD38: a glycoprotein in search of a function. Immunol Today 1994; 15: 95

31 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680

32 Stashenko P, Nadler LM, Hardy R, Schlossman SF. Characterization ofa human B lymphocytespecific antigen. J Immunol1980; 125: 1678

33 Zhong RK. Human blood dendritic cell-like B cells isolated by the 5G9 monoclonal antibody reactive with a novel 220-kDa antigen. J Immunol 1999; 163: 1354

34 Scheringa M, Schraa EO, Bouwman E, et al. Prolongation of survival of guinea pig heart grafts in cobra venom factor-treated rats by splenectomy. No additional effect of cyclosporine. Transplantation 1995; 60: 1350

35 Schmoeckel M, Bhatti FN, Zaidi A, et al. Splenectomy improves survival ofhDAF transgenic pig kidneys in primates. Transplant Proc 1999; 31: 961

36 Watts A, Foley A, Awwad M, et al. Plasma perfusion by apheresis through a Gal immunoaffinity column successfully depletes anti-Gal antibody - experience with 320 aphereses in baboons. Xenotransplantation 2000; 7: 181.

37 Vitetta ES, Stone M, AmIot P, et a!. Phase I immunotoxin trial in patients with B-celllymphoma. Cancer Res 1991; 51 : 4052

38 Zompi S, TuJliez M, Conti F, et al. Rituximab (anti-CD20 monoclonal antibody) for the treatment of patients with clonal Iymphoproliferative disorders after orthotopic liver transplantation: a report of three cases. J Hepatol 2000 Mar; 32: 521

39 Giovino MA, Hawley RJ, Dickerson MW, et al. Xenogeneic bone marrow transplantation: II. Porcine-specific growth factors enhance porcine bone marrow engraftment in an in vitro primate microenvironment. Xenotransplantation 1997; 4: 112

40 Agematsu K, Nagumo H, Oguchi Y, et aL Generation of plasma cells from peripheral blood memory B cells: synergistic effects of interleukin-IO and CD27/CD70 interaction. Blood 1998; 91: 173

41 Kozlowski T, Shimizu A, Lambrigts D, et al. Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption. Transplantation 1999; 67: 18

42 Senderowicz AM, Vitetta E, Headlee D. Complete sustained response of a refractory, posttransplantation, large B-cell lymphoma to an anti-CD22 immunotoxin. Ann Intern Med 1997; 126: 883

43 Slifka MK, Antia R, \Vhitmire JK, et al. Humoral immunity due to long-lived plasma cells. Immunity 1998; 8: 363

44 Manz RA, Thiel A, Radbruch A. Lifetime of plasma cells in the bone marrow. Nature 1997; 388: 133

45 Miller JJ, Cole LJ. The radiation resistance of long-lived lymphocytes and plasma cells in mouse and rat lymph nodes. J ImmunoL 1967; 98: 982

46 Kozlowski T, Monroy R, Xu Y, Glaser R, Awwad M, Cooper DK, Sachs DH. Anti-Ga\(alpha)l-3Gal antibody response to porcine bone marrow in unmodified baboons and baboons conditioned for tolerance induction. Transplantation 1998; 66: 176

47 Ghetie MA, Tucker K, Richardson J, et al. Eradication of minimal disease in severe combined immunodeficient mice with disseminated Daudi lymphoma using chemotherapy and an immunotoxin cocktaiL Blood 1994; 84: 702

48 Flavell OJ, Noss A, Pulford KAF, et al. Systemic therapy with 3BIT, a triple combination coc1.."tail of anti-CDI9, -CD22, and -CD38-saporin immunotoxins, is curative of human B-cell lymphoma in severe combined immunodeficient mice. Cancer Research 1997; 57: 4824

111

Section ll/ - Chapter 4

49 Treon SP, Shima Y, Preffer FI, et al. Treatment of plasma cell dyscrasias by antibody-mediated immunotherapy. Semin Oncol 1999; 26: 97

112

IV Understanding and preventing the thrombotic complications associated with pig-to-baboon xenotransplantation

5 Coagulation and thrombotic disorders associated with xenotransplantation

Adapted from: Alwayn IPJ, Buhler L, Basker M, Goepfert C, Kawai T, Kozlowski T, Ierino F, Sachs DH, Sackstein R, Robson SC, and Cooper DKC. Coagulation! Thrombotic disorders associated with organ and cell xenotransplantation. Transpl. Froc. 2000;32(5):1099. & Buhler L', Basker M', Alwayn IPl, Goepfert C, Kitamura H, Kawai T, Gojo S, Kozlowski T, Ierino FL, Awwad M, Sachs DH, Sackstein R, Robson SC, and Cooper DKC. Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Transplantation 2000;70(9):1323-1331 . • Authors contributed equally

PUBMED-ID

HYPERLINK "/pubmed/10936376"Coagulation/thrombotic disorders associated with organ and cell xenotransplantation. Alwayn IP, Buhler L, Basker M, Goepfert C, Kawai T, Kozlowski T, Ierino F, Sachs DH, Sackstein R, Robson SC, Cooper DK. Transplant Proc. 2000 Aug;32(5):1099. No abstract available. PMID: 10936376 [PubMed - indexed for MEDLINE]

PUBMED-ID

HYPERLINK "/pubmed/11087147"Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Bühler L, Basker M, Alwayn IP, Goepfert C, Kitamura H, Kawai T, Gojo S, Kozlowski T, Ierino FL, Awwad M, Sachs DH, Sackstein R, Robson SC, Cooper DK. Transplantation. 2000 Nov 15;70(9):1323-31. PMID: 11087147 [PubMed - indexed for MEDLINE]

Section IV - Chapter 5

ABSTRACT

Background. Efforts to achieve tolerance to transplanted pig organs In nonhuman

primates by the induction of a state of mixed hematopoietic chimerism have been

associated with disorders of

coagulation and thrombosis. Activation of recipient vascular endothelium and platelets

by porcine hematopoietic cells and/or activation of donor organ vascular endothelium.

Molecular differences between species with respect to regulation of coagulation and

hemostasis may play a role. Irradiation or drug therapy could possibly potentiate

endothelial cell activation and/or injury.

Methods. We have investigated parameters of coagulation and platelet activation in

nonhuman primates following (i) a regimen aimed at inducing mixed hematopoietic

chimerism and tolerance (NMCR that included total body irradiation, T cell depletion

and splenectomy, (ii) pig bone marrow or pig peripheral blood mobilized progenitor cell

transplantation (PCTx, and/or (iii) pig organ transplantation (POTx). Five experimental

groups were studied. Baboons were the recipient subjects in all groups except Group 1.

Group I Cynomolgus monkeys (n~6) underwent NMCR + allotransplantation of

hematopoietic cells and a kidney or heart or NMCR + concordant xenotransplantation

(using baboons as donors) of cells and a kidney; Group 2 Baboons (n~4) underwent

NMCR with or without (+/-) autologous hematopoietic cell infusion; Group 3 (n~12)

PCTx +/- NMCR; Group 4 (n~5) POTx +/- NMCR; Group 5 (n~4) NMCR + PCTx +

POTx. Platelet counts, \\'ith plasma prothrombin time, partial thromboplastin time,

fibrinogen levels, fibrin split products and/or D-dimer were measured.

Results. In the absence of a discordant (porcine) cellular or organ transplant (Groups 1

and 2), NMCR resulted in transient thrombocytopenia only, in keeping with bone

marrow depression from irradiation. PCTx alone (Group 3) was associated with the

rapid development of a tbrombotic thrombocytopenic (TTP)-like microangiopathic state,

that persisted longer when PCTx was combined with NMCR. POTx (+/- NMCR)

(Group 4) was associated with a gradual fall (over several days) in platelet counts and

'fibrinogen with disseminated intravascular coagulation (DIC); after graft excision, the

DIC generally resolved. When NMCR, PCTx and POTx were combined (Group 5), an

116

Coagulation and thrombotic complications

initial TIP-like state was superceded by a consumptive picture of DIC within the first

week, necessitating graft removal.

Conclusions. Both PCTx and POT x lead to profound alterations in hemostasis and

coagulation parameters that must be overcome if discordant xenotransplantation of

hematopoietic cells and organs is to be fully successful. Disordered thromboregulation

could exacerbate vascular damage and potentiate activation of coagulation pathways

following exposure to xenogeneic cells or a vascularized xenograft.

INTRODUCTION

The current shortage of organs for transplantation could be overcome ifpig organs could

be transplanted successfully. However, a number of barriers have to be overcome.

Hyperacute rejection can be prevented by various procedures, such as depletion of anti

pig antibody (1-3) or complement (4-6) in the recipient primate or by the use of organs

from pigs transgenic for one or more human complement regulatory proteins (7,8).

However, even under these circumstances, a delayed fonn of rejection occurs that is

characterized by endothelial cell activation, platelet adhesion and aggregation, with

thrombin and fibrin deposition (9,10). Severe coagulation disturbances have been

documented in nonhuman primates following the transplantation of pig hematopoietic

cells andlor organs (3,11-13, and Buhler, L, et ai, manuscript submitted), Many factors

could contribute to these disturbances, including the therapeutic interventions or

procedures used to suppress the recipient inunune system. Activation of host vascular

endothelium, circulating leukocytes and platelets by infused porcine hematopoietic cells

is an additional possibility. Alternatively, the vascular endothelium within the

transplanted porcine organ may be directly perturbed as a result of the effects of

xenoreactive antibodies (13).

Molecular incompatibilities may also be important factors in both situations (9,10,13-

16). Relevant examples documented include the inability of porcine tissue factor

pathway inhibitor to adequately neutralize human factor Xa, activation of both human

prothrombin and factor X by porcine endothelial cells, the failure of porcine

thrombomodulin to bind human thrombin and hence activate human protein C, and the

117


enhanced potential of porcine von Willebrand factor to associate v.'ith human platelet

GPlb (9-19).

We have reviewed our own data in nonhuman primate auto-, allo-, and concordant xeno

transplantation models and in the miniature swine-to-nonhuman primate discordant

xenotransplantation model in an effort to clarify the relative roles of a regimen aimed to

induce mixed chimerism and tolerance (tolerance-inducing regimen; NMCR), porcine

hematopoietic cell transplantation (PCTx). and porcine organ transplantation (POTx) in

the development of coagulation/thrombotic disturbances. Our observations indicate that

NMCR +1- autologous, allogeneic, or concordant xenogeneic cell or organ

transplantation have minimal disturbances in coagulation. However, in the discordant

combination using comparable NMCR, PCTx results in a thrombotic thrombocytopenic

microangiopathic (TTP)-like state (20, and Biihler, L., et aI, manuscript submitted), and

the development of delayed rejection following POTx leads to disseminated

intravascular coagulation (DIC) (3, II) which progresses unless the organ is excised.

MATERIALS AND METHODS

Animals

Cynomolgus monkeys (Macaca fascicularis) (n~6) of both sexes, weighing 4-6 kg, and

baboons (Papio anubis) (n~24) of both sexes, weighing 10-15 kg (all from Biomedical

Resources Foundation, Houston, TX) were the recipient animals used in these studies.

Cynomolgus monkeys, baboons or Massachusetts General Hospital rvtHC-inbred

miniature swine (Charles River Laboratories, Wilmington, MA), weighing 9-17 kg, were

donors of hematopoietic cells and organs. Cynomolgus monkeys and baboons were of

AB, B or A blood type. All pigs were of 0 blood type.

Care of animals was in accordance wit..t,. the Guide for the Care and Use of Laboratory

Animals prepared by the National Academy of Sciences and published by the National



118


Anesthesia and Surgical Procedures

Sedation, anesthesia, intravenous line placement, splenectomy, porcine kidney and

heterotopic heart transplantation have been described previously in baboons (1,3,21) and

cynomolgus monkeys (22), as have the harvesting and processing of donor bone marrow

(BM) (22,23) and the mobilization and leukapheresis of porcine peripheral blood

progenitor cells (PBPC) (24).

At the time of kidney transplantation, in general one native kidney was excised and the

ureter of the remaining kidney was ligated. In the event ofDIC or rejection developing

within three weeks, necessitating excision of the transplanted kidney, the ligature around

the native ureter was released allowing survival of the recipient.

Conditioning Regimen for Tolerance Induction (NMCR)

The NMCR (See Chapter 1, figure 3) consisted of (i) non-myeloablative whole body

irradiation in either two or three fractions (150 or 100 cGy each, respectively) on days-6

and -5 (total dose 300 cGy); (ii) horse anti-human antithymocyte globulin (ATG) 50

mglkg i.v. daily on days -3, -2 and -1; (iii) thymic irradiation (TI) of700 cGy on day -1;

and (iv) splenectomy on day -8 or 0 (3,20). In baboons undergoing PCTx and/or POTx,

extracorporeal immunoadsorption (EIA) x 3 or 4 was carried out during the previous

week (3,25), and cobra venom factor (approximately 100 units/kg/day) was adntinistered

(and titrated to CH50) throughout the post-transplant course (6,20).

After hematopoietic cell and/or organ transplantation, pharmacologic

immunosuppressive therapy varied slightly between the different experimental groups.

All recipients received cyclosporine (CyA; Novartis, Basel, Switzerland) 10-20

mg/kg/day i.m. from day 0 in cynomolgus monkeys or 15-20 mglkg/day by continuous

i.v. infusion from day -5 in baboons, except where stated to the contrary. A second

agent was added whenever xenotransplantation was being undertaken. This was either

15-deoxyspergualin (Novartis) 6 mglkg/day i.v. on days 0-13 or mycophenolate mofetil

(MMF) 100 mglkg/day i.v. from day -9. Baboons in Groups 3 and 5 also received

methylprednisolone 4 mglkg/day reducing to 0.5 mglkg/day over the first four weeks.

Two baboons receiving autologous BM transplantation received a myeloablative

119


regimen that has been described previously (26). An anti-CD40L mAb (20 mg/kg/i.v.

on alternate days for 8 doses) was substituted for CYA in some baboons in Groups 3-5

(20). There was no direct correlation between the immunosuppressive regimen

administered and the changes seen in coagulation parameters.

Extracorporeal Immunoadsorption (EIA)

When PCTx or POTx was to be undertaken, anti-Galal-3Gal (aGal) antibody was

depleted from the baboon's circulation by the perfusion of blood or plasma through

immunoadsorption colunms containing synthetic Galal-3Galfil-4BGlc-X-Y (aGal type

VI trisaccharide; Alberta Research Council, Edmonton, Alberta, Canada), as described

previously (25).

Supportive Therapy

Kidney transplant recipients were monitored daily by complete blood count, serum

creatinine, blood urea nitrogen, total protein, and electrolytes. In addition, heart

transplant recipients were monitored by daily observation/palpation of the donor heart

contractions. Ofloxacin (50 mg/day) or cefazolin (500 mg/day) was administered daily

as prophylaxis against infection in all baboons of Groups 2-5. Twice weekly blood

cultures were perfonned to detect systemic bacterial or fungal infection. To correct

anemia, thrombocytopenia and coagulopathy, baboon ABO-matched packed red blood

cells, platelets (collected by apheresis or by manual processing), and fresh frozen plasma

(depleted of anti-pig antibody using adsorption colunms or pig red blood cells) were

administered to baboons in Groups 2-5. The indication for blood transfusion was a

hematocrit of <20%, for platelets a platelet count of <lOx !03/mm3 and/or clinical

evidence of bleeding, and for plasma, clinical bleeding or a persistent prothrombin time

of>35 sec.

Measurement of Coagulation Parameters

White blood cell and platelet counts were determined daily by standard methodology

(Excell, Danam Electronics, TX). If the platelet count were <I 00,000/mm3, a blood

smear was prepared, fixed with methanol, air dried, and stained with Wright Giemsa

(Fisher Scientific, Pittsburgh, PA). Platelets were counted under oil immersion.

120


Erythrocyte morphology was also observed. Blood for special assays was collected in

Vacutainer tubes (Becton Dickinson, Rutherford, NJ) containing 0.015 M citrate or

heparin for plasma, and plain tubes for serum. Blood samples were centrifuged at sao g

for 10 min. Plasma and serum samples were immediately stored at -80°C until used in

assays.

Prothrombin time (PT), partial thromboplastin time (PIT) and fibrinogen determinations

were performed on plasma samples at the Hematology Laboratory at the Massachusetts

General Hospital. PT and PTT were assayed by standard methods, and the fibrinogen

measured by the Clauss method (27), using the MDA-lSO automated coagulation

analyzer (Organon Teknika, Durham, NC). Fibrin split products were measured by

standard techniques at the Massachusetts General Hospital laboratory. Ranges of D

dimer were measured using a semiquantitative assay kit, as described in the

manufacturer's instructions (Diagnostica Stago, Asnieres, France), supplemented by

specific enzyme-linked immunosorbent assay (ELISA) (Gold-Dimertest, American

Diagnostica, Greenwich, CT).

Histopathology of Transplanted Organs or Native Organs at Autopsy

Tissues were (i) examined by light microscopy after being formalin-fixed and

stained with hematoxylin and eosin or periodic acid-Schiff and (ii) processed

for immunohistology/fluorescence. To detect platelet aggregates, an indirect

immunoperoxidase technique was applied with a murine monoclonal antibody

directed to human CD62P (P-selectin) (Becton Dickinson, San Jose, CAl. For

v\VF detection, a rabbit polyc1onal antibody to human vWF crossreactive with

baboon vWF was used (DAKO, Glastrup, Denmark). For the detection ofIgG,

IgM, C3 and fibrin deposition, frozen tissue sections were stained using direct

immunofluorescence with FITC-conjugated rabbit polyclonal antibody to

human IgG, IgM, C3 and fibrin (DAKO), all of which were crossreactive with

baboon antigens. Controls consisted of tissue staining with an irrelevant

murine monoclonal antibody or rabbit-anti-human albumin.

121


Experimental Groups

Five experimental groups were studied (Table I).

Group 1 (n~6). Cynomolgus monkeys received NMCR and allo (n~3) or concordant

xeno (n~3) BM cells and kidney (n~2) or heart (n~l) transplants on day O.

Group 2 (n~4). Baboons received either non-myeloablative NMCR alone (n~2) or

myeloablative NMCR and autologous BM transduced -with the porcine al,3

galactosyltransferase gene which was infused intravenously on days 0, I and 2 (n~2).

Group 3 (n~12). PCTx+/-NMCR. Two baboons received pig PBPC (3 x 10 10 cells/kg)

and one received pig BM (14 x 108/kg) without NMCR. Nine baboons received NMCR

followed by PBPC (3 x 101O/kg).

Group 4 (n~4). POTx+/-NMCR. Non-myeloablative NMCR was carried out in one

baboon which received a pig kidney transplant. Three other baboons received kidney

(n~2) or heterotopic heart (n~l) transplants without NMCR.

Group 5 (n~4). NMCR+PCTx+POTx. Following non-myeloablative NMCR, 3 baboons

received kidney transplants and one a heterotopic heart transplant. All received either

PBPC (3 x 101O/kg) (n~l) or pig BM (8-26 x 108/kg) (n~3), the hematopoietic cell

infusion beginning either inunediately after the organ transplant or 24 hours later (Figure

1 ).

RESULTS

The results are summarized in Table 1.

Effects ofNMCR +/- non-discordant cellular/organ Tx (Groups 1 and 2)

Whenever NMCR was administered without PCTx or POTx, i.e. either NMCR alone or

when accompanied by hematopoietic autologous, aIlo or concordant xeno cells and

organs, there was a gradual decrease in platelet numbers over 7-12 days with full

122


recovery to nonnal levels between days 15-18 (Figure I). The thrombocytopenia was

more prolonged following myeloablative NMCR (Group 2) (not shown). However, in all

cases, PT, PTT, fibrinogen, and fibrin split products remained within the normal ranges

or were transiently deranged to a minor extent.

500 .... !:: 400 :::I 0:::-

.:: -2- 300 Q)M _0

J!l ~ 200 <II

1i: 100

O+----.--~._--~--~

-10 n 0 WBlm

10 20 30

ATG Days

Figure 1.

Changes in platelet count in 2 representative recipient baboons from Group 2 that had received NMCR

alone or NMCR and autologous hematopoietic cell transplantation. NMCR alone or NMCR +

autologous cells resulted in a steady fall in platelet count for 10-12 days. with subsequently a rapid

recover)' phase.

Effects of discordant xenogeneic cellular Tx with or without NMCR (pCTx+l-

NMCR) (Group 3)

'Whenever PCTx was carried out, a TIP-like state developed. \¥hen PCTx was carried

out without NMCR, a rapid decrease in platelets (to <20,000 rnrn3) occurred (within 3

days) (Figure 2) with a slight and transient prolongation ofPT «17 sec) and PTT «45

sec) and decrease in fibrinogen level (to 30% of baseline). On blood smear,

schistocytosis was marked (> 12 hpf) and persisted for several weeks. A massive increase

in lactate dehydrogenase (to> 10,000 units) was seen. Fibrin split products and D-dimer

123

Section IV-Chapter 5

increased to high levels but rapidly nonnalized. Platelets recovered within 5-9 days and

fibrinogen by day 15. In one baboon, after recovery of coagulation parameters, a second

set ofPBPCs was infused, resulting in similar changes.

When NMCR preceded PCTx, there was a rapid and profound fall in platelet count (to

<20 rom3) following the first infusion of porcine cells (even after as few cells as 1 x

10(/kg) (Figure 2). Tbrombocytopenia persisted for approximately two weeks, during

which platelet transfusions were required. Thereafter, recovery of platelet count was

relatively rapid. The initial rapid onset of thrombocytopenia was temporally associated

with the PCTx. In contrast to when PCTx was carried out alone, the delayed recovery

was associated with the effects of myelosuppression brought about by NMCR.

Schistocytosis was again marked (>12 hpf) unless prophylactic therapy was given to

reduce platelet and endothelial cell activation (20) and lactate dehydrogenase rose. In

some baboons there was mild purpura, transient neurologic disorder (transient collapse,

lethargy), or transient renal dysfunction (in one baboon).

-!: ::J

750

8 :;, 500 a;~ _0

.e~ .l!! 250 0..

~PCTxalone

-PCTx+NMCR

O+-~~~~-.---.---, -10 nO

WBlm

10 20 30 40

ATG Days

Figure 2.

Changes in platelet count in 2 representative recipient baboons in Group 3 that had received pig

hematopoietic cells alone or following NMCR. The infusion of pig hematopoietic cells resulted in an

immediate thrombocytopenia, which recovered quickly unless NMCR had also been administered and

bone marrow suppression had been induced.

124


One of two baboons that received PBPC alone died after receiving a second infusion of

PBPC. After the second PBPC infusion, severe thrombocytopenia developed, with an

increase in LDH to >25,000 unitslL. PT, PTT and fibrinogen level remained relatively

stable. The baboon died with features of cardiorespiratory failure within 4 days. Autopsy

showed gross hemorrhage in the lungs and heart and in other essential organs.

Histological examination confinned interstitial hemorrhage, particularly in the heart, the

lungs, kidneys, and adrenal glands. Immunohistochemistry showed deposition of

platelets in small vessels, expression of vWF on the capillary walls, with fibrin

deposition (data not shown).

TABLE 1

EXPERIMENTAL GROUPS AND SUMMARY OF OBSERVED

CHANGES IN COAGULATION PARAMETERS

GROUPS REGIMEI' PLATELETS ..IT pn HBRNOGEN

I (n"'6) l';1v!CR + allo or

concordant ~

xeno HCs and

organ Tx

2(nooo4} NMCR +/. auto-

logous HCs "I<~

3 (n"'12) PCTx +/- NMCR ~~ ,

4 (n"'5) POTx +/- !\MCR " 1'1'

5 (n=1) NMCR +PCTx h~ H, ttt +POTx

?'-lMCR '" tolerance-inducing regimen

HCs '" hematopoietic cells (bone marrow or peripheral blood mobilized progenitor cells)

PCTx '" porcine hematopoietic cell transplantation

POTx = porcine organ transplantation

Change in parameter

Decrease ... mild, ... t modemte, Ht profound

Increase l' mild, 1'1' moderate, t1t profound

t

~

H

,H

D-DTME.R

,

t

tt

125


One baboon that received NMCR + PBPC died from acute cardiorespiratory failure on

day 5 with an undetectable level of circulating platelets, despite platelet transfusion.

There was a transient increase in PI to twice the baseline level, but this persisted for

only 1-2 days before nonnalizing. PTT was not significantly altered. The fibrinogen

level fell modestly 4-6 days after the initial PCTx. D-dimer levels increased between

days 1-7, but again normalized rapidly. At autopsy, the changes were similar to those

above. In addition, however, there was massive hemorrhage in all mesenteric lymph

nodes.

Effects of discordant xenogeneic organ Tx with or without NMCR (pOTx+/

NMCR) (Group 4)

'Whenever POIx was performed, an apparent consumptive coagulopathy and DIC

occlllTed conclllTently with the development of delayed antibody-mediated rejection.

When POTx was performed +/- NMCR, gradual but steady falls in both platelet count

and fibrinogen level occlllTed. The first indication that DIe was developing was

frequently an increase in fibrinolytic activity indicated by a rise in fibrin split products or

D-dimer (Table 2).

After 1-3 days, in 4 of the 5 cases there was a precipitate rise in PT (data not shown).

Clinical bleeding occurred in 2 baboons with hematuria, and both died on day 9 with

massive intraperitoneal hemorrhage. Rejected xenografts revealed microthrombi in

glomeruli and microvasculature with hemorrhagic and necrotic ureters, particularly in

the distal portion; sequential deposits of platelets (platelet microthrombi) and fibrin were

also seen (data not shown). The changes of DIe resolved only in those baboons where

the transplanted porcine organ was excised as an emergency procedure (Figure 3, and

Table 2). Histopathological examination of the excised organ demonstrated variable

features of rejection with sometimes only 20% of the organ affected by interstitial and

deposition of platelets and fibrin. (Fibrin deposition was not seen in native organs,

indicating a localised response.) IgM deposition was seen in the grafted organ at the time

of excision, but IgG deposition was variable (sometimes absent or minimal) depending

126


on the pre and post-transplant therapy given. Complement deposition was frequently

absent as a resuly of therapy with cobra venom factor.

TABLE 2

D-DIMER LEVELS IN TWO GROUP 4 BABOONS

FOLLOWING PIG KIDNEY TRANSPLANTATION

DAY 875-18 875-34

Pre POTx 0 0.5

Post-POTx

<2 2-8

2 2-8 0.5-2

5 2-8 2-8

AtGTx >8 >8

48h post-GTx 0.5 0.5

POTx = porcine organ transplantation

GTx = graftectomy

Effects of combined discordant xenogeneic cellular and organ Tx with NMCR

(NMCR+PCTx+POTx) (Group 5)

When NMCR was combined with both PCTx and POTx, all baboons developed

immediate and profound thrombocytopenia following the first PCTx, which extended for

12-15 days (Figure 3), and required a greater number of platelet transfusions compared

to Group 3. This was followed by a progressive fall in fibrinogen and a prolongation of

PT, indicating consumption of coagulation factors such as seen in DIe. As with POTx

alone, these coagulation parameters resolved only if grafiectomy was perfonned.

All of the transplanted organs had to be removed before day 15 to prevent worsening

DIe. Histopathological examination of the rejected organ, however, again showed

considerable variability in the extent of immtme injury, with patchy localized injury in

some cases. At excision of the graft, there was variable IgM, IgG and complement

deposition as in Group 4. When NMCR was followed by both PCTx and POTx,

127


therefore, the features seen were a combination of the thrombotic microangiopathy that

occurred following PCTx alone and the consumptive state that occurred following POTx

alone.

~ Fibrinogen (mg/dl)

500

c: 400 "'-8':E 300 c: C)

~.s 200 u.

100

Grafiectomy

~ Platelet count

00

00 "'C Ai

~ -.... CD 300 ~~ -1= 0 200.::::: g

::::I 100 -

O+----r~~~--~--~~ -10 10 20 30

Days

Figure 3.

Changes in platelet count and plasma fibrinogen concentration in a representative recipient baboon from

Group 5 (NMCR+PCTx+POTx). When both pig hematopoietic cel!s and a kidney were transplanted in

combination in a baboon which had also received NMCR, there was an immediate thrombocytopenia

which persisted for 14 days until the effects of myelosuppression had worn off. There was also a steady

reduction in fibrinogen until the pig organ was excised (graftectomy), following which there was rapid

recovery.

DISCUSSION

We have presented evidence for profound perturbations in hemostasis, coagulation and

thromboregulation that occur consistently with the transplantation of discordant

xenogeneic cells and/or organs in the the miniature swine-to-nonhuman primate modeL

The coagulopathies observed after discordant organ xenotransplantation in this model

were first documented by lerino (II) and Kozlowski (3) with their respective colleagues,

and have also been described in the pig-to-baboon model by Meyer et al (12), The

present study, which includes a small number of the experiments reported by lerino and

128


Kozlowski (3, II ,26), contributes additional information on this topic and further

elucidates this important consequence of discordant xenograft rejection.

In particular, we demonstrate that NMCR alone or when combined with autologous, alto

or xeno hematopoietic cell transplantation +/- organ allo- or concordant xeno

transplantation resulted in relatively minimal and transient changes in coagulation

parameters of the recipient animal (either cynomolgus monkey or baboon). The NMCR

utilized at our center, therefore, although prolonging the duration of thrombocytopenia

following PCIx, would not otherwise appear to playa direct role in the development of

either the thrombotic angiopathic state seen following PCTx or the consumptive

coagulopathy seen after POIx. However, it is possible that whole body irradiation could

'sensitize' the vascular endothelium making it more susceptible to activation by PBPC.

PCIx, whether alone or in combination with NMCR, caused a profound

thrombocytopenia, limiting the number of porcine cells that could be transfused into the

recipient baboon. In the absence ofNMCR, recovery of platelet count following PCTx

occurred within 5-9 days. The myelosuppression caused by NMCR delayed recovery of

platelets for approximately two weeks. A TTP-like state developed, which has been

investigated further and will be fully described elsewhere (Btihler, L., et a1. manuscript

submitted). In briet~ animals rapidly became thrombocytopenic, developed schistocytes

and demonstrated features of microangiopathy, associated \\ith a rise in lactate

dehydrogenase. Only minor fluctuations in plasma levels of von Willebrand factor were

seen, with transient shifts in von Willebrand factor multimeric patterns. No changes in

von Willebrand factor-cleaving protease activity were noted (data not shown). TTP can

progress to a state where widespread organ damage can trigger release of thromboplastin

and a consumptive coagulopathy (DIC); this was not a feature in the present studies

except under agonal circumstances in 2 baboons in Group 3.

This TTP-like state is therefore comparable to that seen complicating autologous or

allogeneic hematopoietic stem cell transplantation in humans (28). As this complication

was seen in baboons receiving cobra venom factor (as well as in baboons receiving

hematopoietic cells taken from pigs transgenic for human decay accelerating factor -

129


data not shown), this indicates that depletion of complement factors does not preclude

the TTP-like state.

We are currently investigating the mechanisms whereby PBPC activate primate

endothelial cells, lenkocytes and/or platelets. Many of the natural anticoagulants and

complement regulators are expressed by endothelial cells and monocyte-macrophages.

These anticoagulant proteins are largely ineffective across discordant xenogeneic species

barriers (19). Such molecular incompatibilities may lead to activation of coagulation

following the infusion of xenogeneic leukocytes and could result in (i) excessive fibrin

deposition upon the infused porcine cells with consequent platelet interactions mediated

via GPIIbIIIa or (ii) direct upregulation of adhesive proteins with cellular activation,

predisposing to lenkocyte-platelet aggregates.

Some of the baboons being studied, particularly in Groups 3 and 5, received therapy

aimed at reducing the activation of the vascular endothelium and platelets of the host.

This therapy (consisting of prostacyclin, heparin and methylprednisolone) had some

beneficial effect in reducing the features of the TIP-like state, particularly in reducing

the need for platelet transfusion (data not shown), but was not completely successful

(20).

The manifestations of TTP were quite different from the widespread coagulation

changes seen when POTx was performed. Here coagulation-factor consumption and DIC

developed, generally within 1-2 weeks and concurrent with the development of

xenograft rejection. This was exemplified by a steady fall in fibrinogen and platelets and

a late rise in PT. In some cases, after several days, PT rose acutely to a high level,

followed by clinical hemorrhage leading to death of the recipient unless the graft was

removed as an emergency. Following graftectomy, the coagulopathic state generally,

but not always, rapidly resolved. "When both PCTx and POTx were combined, an initial

TTP-like state was seen, which was exacerbated by the development of xenograft

rejection and the evolution of DIe. The consumptive coagulopathy then became the

predominant feature.

130


Of significance was the fact that when graftectomy was necessitated by a rapidly

deteriorating coagulopathic state, on occasion the transplanted porcine organ looked

relatively nonnal macroscopically, and histological examination showed only moderate

immune injury. The extent of histopathological change in the fonn of interstitial

hemorrhage or fibrin deposition could involve as little as 20% of the organ. We suggest,

therefore, that the DIC may not necessarily be a result of the organ damage linked to the

acute vascular rejection that is developing, but may be associated with factors common

to both the development of coagulation disturbances and acute vascular rejection, such

as vascular endothelial cell activation.

Quiescent endothelial cells express potent thromboregula~ory factors, including the

vascular ATP-diphosphohydrolase (or CD39), tissue factor pathway inhibitor, and

thrombomodulin (29). In addition to heparan sulphate (30), these proteins are lost in

association with xenograft rejection. Hence the activated endothelial cell becomes

thrombophilic, and may initiate and perpetuate the thrombotic reactions that result in

systemic depletion of coagulation factors seen in DIC (9,10,18,29). Tissue factor is an

important initiating factor of coagulation and is expressed by activated endothelial

cells and denuded vasculature. In vitro assays have ShO\\l1 that porcine endothelial

cells stimulated by human serum are able to increase tissue factor via IL-l a.

production and require the presence of sub-lytic complement Membrane Attack

Complex (MAC) components (31). Despite cobra venom factor therapy in our POTx

experiments, such sub-lytic complement activation is still likely and may have

induced increased expression of tissue factor on the activated endothelium of the

xenografts. In addition, porcine endothelial ceBs, including hDAF transgenic cells,

have the capacity to upregulate tissue factor following exposure to other inflammatory

mediators, e.g. twnor necrosis factor released by infiltrating mononuclear cells (32).

The development of DIC in these xenografts has parallels to the consumptive

coagulopathy seen with the Shwartzman phenomenon in antibody-mediated vascular

injury of allografts (33). Removal of the activated endothelium (with the graft) leads to a

greater response to therapy in the fonn of fresh frozen plasma and platelet transfusions.

131


We are not alone in identifying DIC following xenotransplantation experiments. This

was first described in the 1960s (34,35), although there have been few reports in the

pig-to-nonhuman primate model. In the pig-to-baboon renal transplant model, Meyer

et al. (12) have also shown evidence for platelet activation, formation of thrombin

antithrombin III complexes, a fall in fibrinogen, and an increase in fibrinolytic

activity, all suggesting DIe. They also demonstrated that the transplantation of organs

from pigs with homozygous VOn Willebrand's disease, that were severely deficient in

von Willebrand factor, did not prevent platelet and coagulation activation. None of

their animals, either control or with von Willebrand disease, presented with overt

bleeding. d'Apice's group in Australia has recently observed a DIC-like state in

unmodified (non-immunosuppressed) baboons receiving pig kidneys transgenic for

human CD55 and CD59 (36). Other groups have not reported major coagulation

disorders following pig-to-nonhuman primate organ transplantation. Indeed, it was

not observed by the Cape Town and Oklahoma groups of which one of us (DKCC)

was a member (6,37,38).

Numerous potential differences in experimental protocols may accotu1t for this

discrepancy, including, for example, differences in (i) the species of nonhuman

primate used as recipient (e.g. cynomolgus monkey or baboon), (ii) the breed of

organ-donor pig (e.g. large white or miniature swine), (iii) whether the pig has been

genetically modified or not (e.g. the presence of hDAF on its vascular endothelium),

(iv) the organ transplanted (kidney or heart), (v) the immunosuppressive regimen

(where there are numerous variables, including alkylating agents at different dosages),

and (vi) any adjunctive therapy administered (e.g. the concomitant infusion of

hematopoietic cells or the intravenous administration of prostacyclin). However, we

have observed DIC both in cynomolgus monkeys and baboons, after the

transplantation of pig kidneys and hearts, when induction therapy was with whole

body irradiation or with cyclophosphamide, and with and without cobra venom factor.

Apart from the report from d' Apice's group mentioned above, DIC does not seem to

have been observed by those using organs fTom pigs transgenic for one or more

complement regulatory proteins (7,8,39). However, we have recently seen this

complication in 2 of 3 baboons receiving hDAF kidneys at our center (Buhler, L., et

132


aI, manuscript In preparation); all 3 had received induction therapy with

cyclophosphamide and not whole body irradiation. As we routinely adsorb out anti

Gal antibody before the transplant, it is possible that it is the slow retwn of antibody,

with gradual binding to the graft vascular endothelium (rather than immediate and

massive antibody binding as occurs when antibody is not removed), that is important

in the development of a procoagulant state. Furthermore, procedures that deplete

antibody may also deplete or perturb labile coagulation factors. Others, however, have

included antibody adsorption in their protocols (by similar but not identical

techniques) but have not reported consumptive coagulopathy as a complication

(39,40). It is therefore difficult to explain why this complication has been observed

frequently at some laboratories but not at other centers using similar or even identical

protocols.

We conclude that both PCTx and POTx result in significant changes in coagulation

that must be further investigated and overcome if discordant xenotransplantation of

hematopoietic cells and organs is to be fully successful. The TTP-like state that

follows PCTx limits the number of pig hematopoietic cells that can be infused and

therefore may be a factor in the extent of pig cell chimerism that can be obtained.

Under the circumstances of the experiments reported here, rejection of a porcine

organ xenotransplant was consistently associated with the development of DIC,

necessitating excision of the transplanted organ, whether it were a kidney or a heart.

Importantly, in some cases, DIC developed before there were widespread changes of

inununological injury to the transplanted organ.

DIC might therefore be considered to be a surrogate marker for developing antibody

mediated rejection. Our observations, and those of others, suggest that the discordant

xenogeneic vasculature may be predisposed lUlder certain circumstances to initiate

thrombotic reactions in vivo.

133


REFERENCES

1. Sablinski T, Latinne 0, Gianello P, et al. Xenotransplantation of pig kidneys to nonhuman primates. I. Development of the model. XenotTansplantation 1995; 2: 264

2. Cooper DKC, Cairns TDH, Taube DH. Extracorporeal immunoadsorption of anti-pig antibody in baboons using aGal oligosaccharide immunoaffinity columns. Xeno 1996; 4: 27

3. Kozlowski T, Shimizu A, Lambrigts 0, et al. Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption. Transplantation 1999; 67: 18

4. Leventhal JR, Dalmasso AP, Cromwell JW, et aI. Prolongation of cardiac xenograft survival by depletion of complement. Transplantation 1993; 55: 857

5. Pruitt SK, Kirk AD, Bollinger RR, et al. The effect of soluble complement receptor type I on hyperacute rejection of porcine xenografis. Transplantation 1994; 57: 363

6. Kobayashi T, Taniguchi S, Neethling FA, et al. Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy. Transplantation 1997; 64: 1255

7. Schmoeckel M, Bhatti FNK, Zaidi A., et al. Orthotopic heart transplantation in a transgenic pig-toprimate model. Transplantation 1998; 65: 1570

8. Zaidi A, Scmoeckel M, Bhatti FNK, et aJ. Life-supporting pig~to~primate renal xenotransplantation using genetically modified donors. Transplantation 1998; 65: J 584

9. Bach FH, Robson SC, Ferran C, et al. Endothelial cell activation and thromboregulation during xenograft rejection (review). Immunol Rev 1994; 141: 5

10. Bach FH, Winkler H, Derran C, et al. Delayed xenograft rejection. Immuno! Today 1996; 17: 379 11. lerino FL, Kozlowski T, Siegel 18, et a!. Disseminated intravascular coagulation in association with

the delayed rejection of pig-to-baboon renal xenografts. Transplantation 1998; 66: 1439 12. Meyer C, Wolf P, Romain N, et al. Use of von Willebrand diseased kidney as donor in a pig-to

primate model ofxenotransplantation. Transplantation 1999; 67: 38 13. Robson SC, Schulte am Esch II J, Bach FH. Factors in xenograft rejection. Ann NY Acad Sci 1999;

875:261 14. Jurd KM, Gibbs RV, Hunt BJ. Activation of human prothrombin by porcine aortic endothelial cells

a potential barrier to pig-to-human xenotransplantation. Blood Coagul Fibrinolysis 1996; 7: 336 15. Jurd KM, Lee J, Cairns T, et a!. Effect ofGalal,3GaIf31,4 G!cNAc and complement depletion on

haemostatic activation in an in vitro model of the pig-to-human xenograft reaction. Transplant Proc 1996;28:637

16. Siegel JB, Grey ST, Lesnikoski BA, et al. Xenogeneic endothelial cells activate human prothrombin. Transplantation 1997; 64: 888

17. Kopp CW, Siegel JB, Hancock WW, et al. Effect of porcine endothelial tissue factor pathway inhibitor on human coagulation factors. Transplantation 1997; 63:749

18. Lawson JH, Daniels LJ, Platt JL. The evaluation of thrombomodulin activity in porcine to human xenotransplantation. Transplant ProC 1997; 29: 884

19. Schulte J, Siegel JB, Bach FH, Robson SC. The A 1 domain of von Willebrand factor expressed on cell membranes directly activates platelets. Blood 1996; 88(Suppl): 81

20. Buhler L, Awwad M, Basker M, et al. High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response. Transplantation. Accepted for publication.

20. Cooper DKC, Ye Y, Niekrasz M. Heart transplantation in primates. In: Handbook of Animal Models in Transplantation Research. Cramer DV, Podesta L, Makowka L (eds). Boca Raton, CRC, 1993, p. 173

21. Kawai T, Cosimi B, Colvin R, et al. Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 1995; 59: 2561

23. Pennington LR, Sakamoto K, Popitz-Bergez FA, et al. Bone marrow transplantation in miniattrre swine. I. Development of the model. Transplantation 1988; 45: 21

24. Nash K, Chang Q, Watts A., et a1. Peripheral blood progenitor cell mobilization and leukapheresis in pigs. Lab Anim Sci. 1999; 49: 645

25. Kozlowski T, lerino FL, Lambrigts 0, et a1. Depletion ofanti-Galal-3Gal antibody in baboons by specific a-Gal immunoaffinity columns. Xenotransplantation 1998; 5: 122

26. lerino FL, Gojo S, Banerjee PT, et at. Transfer of swine major histocompatibility complex Class II

134


genes into autologous bone marrow cells of baboons for the induction of tolerance across xenogeneic barriers. Transplantation 1999; 67: 1119

27. Clauss A. Rapid physiological coagulation method in the detennination of fibrinogen. Acta Hematol (Basel) 1957; 17: 237

28. Pettitt AR, Clark RE. Thrombotic microangiopathy following bone marrow transplantation. Bone Marrow Transplant 1994; 14: 495

29. Robson SC, Kopp C. Disordered thromboregulation in discordant xenograft rejection. Life Sci 1996; 6: 34

30. Platt JL, VercelJoti GM, Lindman BJ, et al. Release of hepar an sulphate from endothelial cells. J.Exp.Med. 1990; 171:1363.

31. Saadi S, Holzknecht RA, Patte PP, et al. Complement-mediated regulation of tissue factor activity in endothelium. J Exp Med 1995; 182: 1807

32. Tucker A W, Carrington CA, Richards AC et al. Endothelial cells from human decay acceleration factor transgenic pigs are protected against complement mediated tissue factor expression in vitro. Transplant Proc 1997; 29; 888

33. Starzl TE, Boehmig HJ, Amemiya H, et a1. Clotting changes, including disseminated intravascular coagulation, during rapid renal homograft rejection. N Engl J.Med 1970; 278: 642

34. Rosenberg JC, Broersma RJ, Bullemer G, et al. Relationship of platelets, blood coagulation, and fibrinolysis to hyperacute rejection of renal xenografts. Transplantation 1969; 8: 152

35. Broersma RJ, Bullemer GD, Rosenberg JC, et al. Coagulation changes in hyperacute rejection of renal xenografts. Thromb Diath Haem.-Supplementum 1969; 36: 333

36. Cowan P, Goodman D, Aminian A, et a!. Disseminated intravascular coagulation in nooimmunosuppressed baboons receiving transgenic pig renal xenografts. (Abstract 0185). 5th Congress of the International Xenotransplantation Association, Nagoya, 1999

37. Cooper DKC, Human PA, Lexer G, et al. Effects ofcyc1osporine and antibody adsorption on pig cardiac xenograft survival in the baboon. J Heart Transplant 1988; 7: 238

38. Cooper DKC, Cairns TDH, Taube DB. Extracorporeal inununoadsorption of aGal antibodies. Xeno 1996;427

39. Lin SS, Weidner BC, Byrne GW et al. The role of antibodies in acute vascular rejection of pig-tobaboon cardiac transplants. J Clin Invest 1998; 101: 1745

40. Leventhal JR, Sakiyalak P, Witson J, et al. The synergistic effect of combined antibody and complement depletion on discordant cardiac xenograft survival in nonhuman primates. Transplantation 1994; 57: 974

135


136

6 Mechanisms of thrombocytopenia following xenogeneic hematopoietic progenitor cell transplantation

Adapted from: Alwayn IPl, Buhler L, Appel JZ Ill, Csizmadia E, Harper D, Down 1, Awwad M, Kitamura H, Sackstein R, Cooper DKC·, and Robson SC·. Mechanisms of thrombocytopenia following x£.flogeneic hematopoietic progenitor cell transplantation. Transplantation 2001. In press . • Joint senior authors


ABSTRACT

Introduction. Attempts to induce tolerance though mixed hematopoietic chimerism

in the discordant pig-to-baboon xenotransplantation model are complicated by a

potentially fatal thrombotic microangiopathy in the recipient baboons. This state

develops inunediately following the infusion of porcine mobilized peripheral blood

leukocytes, containing progenitor cells (PBPC). In the present study, we examined the

interaction of infused porcine PBPC with recipient platelets in vivo in baboons and

investigated the underlying mechanisms using an in vitro model.

Methods. Two naIve baboons and six baboons pre-conditioned with irradiation and

inununosuppression that received porcine PBPC were evaluated in vivo. The

interaction of porcine and baboon PBPC with baboon platelets was investigated by an

in vitro platelet aggregation assay. Fresh and cryopreserved PBPC were evaluated as

well as PBPC obtained from growth-factor-mobilized and unmobilized pigs.

Furthenuore, cellular subsets of PBPC were assessed for potential to induce platelet

aggregation. Inununohistochemical staining was performed on platelet-leukocyte

aggregates and potential inhibition of aggregation with anti-P-selectin and anti

CDIS4 mAbs, or eptifibatide (a GPIIb/IlIa receptor antagonist), was tested.

Results. All baboons that received porcine PBPC rapidly developed marked

tbrombocytopenia «20,OOO/).lI), elevated serum lactate dehydrogenase (>l,SOOU/I),

schistocytosis, and platelet aggregates on blood smear. Three baboons died (two

untreated and one pre-conditioned), and substantive platelet aggregates containing

porcine leukocytes were observed in the microvasculature of lungs and kidneys. h!

vitro, porcine, but not baboon, PBPC induced aggregation of baboon platelets in a

dose-dependent manner. lnununohistological examination of these aggregates

confinued the incorporation of porcine leukocytes. Cryopreserved PBPC caused less

aggregation than fresh PBPC, and growth-factor-mobilized PBPC induced less

aggregation than unmobilized PBPC. Aggregation was fully abrogated by the addition

of eptifibatide, and modulated by anti-P-selectin and anti-CD1S4 monoclonal

antibodies that recognize adhesion receptors on activated platelets. Purified fractions

(granulocytes, CD2+, and CD2" cells) of porcine PBPC did not initiate aggregation,

138

Mechanisms o/thrombocytopenia

whereas addition of exogenous porcine PBPC membranes (erythrocytes, dead cells,

and/or platelets) to the purified fractions exacerbated the aggregation response.

Conclusions. These data indicate that porcine PBPC mediate aggregation of baboon

platelets. This process likely contributes to the thrombotic microangiopathy observed

following PBPC transplantation in the pig-to-baboon model. Eptifibatide can fully

abrogate platelet aggregation induced by porcine PBPC in vitro. Purification of the

precursors from porcine PBPC and/or treatment of baboons with eptifibatide may be

beneficial.

INTRODUCTION

The use of porcine xenografts as an alternative for human cadaveric organs is viewed

by many as a potential solution to the increasing shortage of allografts (1,2). Although

many advances have recently been made with respect to understanding the

immunological basis of rejection of highly disparate organs when transplanted into

nonhuman primates, xenografts generally do not survive beyond approximately one

month (3,4). This process is, at least in part, related to host immunological responses

to the xenograft.

In our efforts to induce tolerance to porcine organs transplanted to nonhuman

primates, we have attempted to induce a state of mixed hematopoietic chimerism (5).

Such an approach has been successful, without· major complications, in rodent

allogeneic and xenogeneic transplantation models, and also in swine and primate

allogeneic models. In these models, immunological tolerance is associated with

indefinite survival of the grafts (6, 7, 8, 9). When extending this approach to the

discordant pig-to-baboon model, using large numbers of mobilized porcine

leukocytes, containing approximately 2% progenitor cells (PBPC), however, a

thrombotic microangiopathy has been observed (10). This phenomenon did not occur

when smaller numbers of porcine bone marrow cells were transplanted into baboons

(11) and, importantly, when a comparable number of cells were transplanted in a

swine allotransplantation model (8). The clinicopathological entity included

neurological and renal disturbances with fever, immediate thrombocytopenia,

139


markedly elevated serum lactate dehydrogenase levels, schistocytosis on blood smear,

with microvascular thrombosis of lungs, heart, and IGdneys on autopsy (12).

Thrombotic microangiopathy has also been observed following clinical autologous

and allogeneic bone marrow transplantation. Here the underlying pathophysiology is

thought to be widespread endothelial cell damage associated with several factors,

including inflammation, cyclosporine, or graft-versus-host disease (13). This state,

however, does not generally develop before the transplanted marrow has engrafted,

whereas the thrombotic microangiopathy in our studies developed rapidly after

porcine PBPC (PPBPC) transplantation. We therefore postulated that the etiology of

these two pathologic states may differ, in that porcine leukocytes may be directly

interacting with baboon platelets in vivo. This process may be accentuated by the

existence of molecular incompatibilities that would facilitate these interactions (14).

We have therefore evaluated such interactions in pre-conditioned and unconditioned

baboons receiving large numbers ofpPBPC (1-3xlO IO cells/kg) in vivo, and correlated

these observations with pPBPC-platelet interactions in vitro. Furthermore, several

agents aimed at preventing these interactions were studied in vitro. Effects of these

individual components of the conditioning regimen on platelet aggregation are

discussed elsewhere (Appel JZ III, et aI., manuscript in preparation).

MATERIAL AND METHODS

In vivo experiments

Animals

Naive baboons (Papio anubis, n=8, Biomedical Resources Foundation, Houston, TX),

of known ABO blood group and weighing 9-15 kg, were used as recipients ofpPBPC,

as donors of platelet-rich plasma (PRP), or as donors of baboon PBPC.

Massachusetts General Hospital MHC-inbred miniature swine (n=6, Charles River

Laboratories, Wilmington, MA), of blood group 0 and weighing 18-40 kg, served as

donors of pPBPC. All experiments were performed in accordance with the Guide for

the Care and Use of Laboratory Animals prepared by the National Academy of

140

Mechanisms of thrombocytopenia

Sciences and published by the National Institutes of Health. Protocols were approved

by the Massachusetts General Hospital Subcommittee on Research Animal Care.

Surgical procedures

Details of anesthesia, intravenous (iv) catheter placement in pigs and baboons, and

intra-arterial line placement, splenectomy, and extracorporeal immunoadsorptions of

anti-aGal antibodies in baboons have been described elsewhere (15, 16, 17).

Non-myeloablative conditioning regimen (NMCR)

This regimen has been described previously (18) and consists of -induction therapy

with splenectomy, whole body irradiation, thymic irradiation, anti-thymocyte

globulin, and multiple extracorporeal immunoadsorptions of anti-a Gal antibodies.

Maintenance therapy consists of anti-CD154 monoclonal antibody (mAb) +1-

cyclosporine, mycophenolate mofetil, cobra venom factor, prost acyl in, heparin, and

methylprednisolone. Porcine growth factors (IL-3 and stem cell factor) were

administered to promote pig cell engraftment.

Mobilization and leukapheresis of P BPC in pigs and baboon

This procedure was performed as previously described (19). In summary, pigs were

treated with recombinant hematopoietic growth factors before and during the period

of collection of PBPC. Leukapheresis was carried out on days 5-9 and 12-16

following commencement of growth factor mobilization using a Cobe Spectra

apheresis machine (Cobe, Lakewood, CO). Baboons were treated with recombinant

hematopoietic growth factors (as for the pigs) and leukapheresis was carried out on

day 7 alone. Plasma was removed from the product by centrifugation at 920 g for 7

minutes. The collected cells were washed and used for in vitro assays, or frozen and

stored in 5% DMSO until transplantation or until used for in vitro assays.

Preparation of pPBPCfor transplantation

Cryopreserved PBPC were thawed by immersion in a 37 - 40° C water bath with

gentle agitation for 1 - 2 minutes, washed twice with calcium-, magnesium, and

phenol red-free Hanks' balanced salt solution (HBSS. Mediatech, Herndon, VA), and

141

Section 1 V - Chapter 6

resuspended in the same solution for immediate transplantation to the recipient

baboon. Viability was assessed with trypan blue staining.

Hematologic and serum chemistry parameters

Daily blood samples were taken for measurement of platelet count (Excell, Danam

Electronics, Dallas, TX), and prothrombin time (PT), partial thromboplastin time

(PTT), fibrinogen, and fibrinogen degradation products (Hematology Laboratory,

Massachusetts General Hospital). Blood smears were also prepared on a daily basis,

fixed with methanol, air dried, and stained with Wright Giemsa (Fisher Scientific,

Pittsburgh, PA). Platelets were counted under oil immersion, and erythrocyte

morphology was observed. Daily serum samples were obtained for the measurement

of lactate dehydrogenase levels (Chemistry Laboratory, Massachusetts General

Hospital).

Immunopathology

Tissues were (i) formalin-fixed, stained with hematoxylin and eosin for evaluation by

light microscopy, or (ii) processed for immunohistology using standard methodology.

Baboon platelets and porcme cells were detected by standard indirect

irnmunoperoxidase technique, using the murine anti-CD62 (P-selectin) mAb cross

reactive with baboon (Becton Dickinson, San Jose, CA) and the porcine pan

leukocyte mAb (I 030H-I-19), respectively.


Preparation of platelet-rich plasma (P RP) and platelet-poor plasma

Blood was drawn from naIve baboons (lO mI), citrated, and centrifuged at 280 g for

15 minutes at 22°C. The supernatant, consisting of PRP containing approximately

lxl08 platelets/ml (as measured with a Coulter counter, Beckman Coulter, Inc.,

Fullerton, CA), was transferred in 400 ).11 aliquots to siliconized glass cuvettes and

used as experimental samples. The remaining pellet was further centrifuged at 2,400g

for 2 minutes at 22°C, the resulting supernatant, platelet-poor plasma (containing less

than Ixl03 platelets/ml), was transferred in a 400 fll aliquot to a siliconized glass

cuvette and used as reference sample.

142

Mechanisms a/thrombocytopenia

Preparation of peripheral blood progenitor cells (PEPC)

Fresh pPBPC, and frozen baboon or pPBPC, were processed as described above. The

cells were resuspended in HBSS to reach a final concentration of approximately I xl 04

cells/fll.

Cell sorting

A MoFlo Cytomation High Speed Cell Sorter (Cytomation, Fort Collins, CO) was

used to purify and sort fractions of fresh or cryopreserved pPBPC. The product was

washed twice in phosphate buffered saline containing 2% fetal bovine serum, but

without calcium or magnesium, and lx108 cells were resuspended at lx107/ml. The

cells were then incubated with the murine biotinylated anti-porcine CD2 (MSA4)

mAb, and subsequently labeled with streptavidin phycoerythrin, Cells were then

washed, resuspended at 5xl07/ml and run through the cell sorter. The pPBPC product

was sorted for CD2+ and CD2- cells based on lymphocyte and fluorescence gating.

Sorting of granulocytes and exogenous membranes (debris, consisting of erythrocytes,

platelets, or dead cells) was based on forward-side scatter. Approximately 2xl06

CD2+ and CDT cells, 2xl05 granulocytes, and 2xl06 exogenous membrane fragments

(as measured per event) were used for each assay. Data acquisition and analysis were

performed with Cyclops Summit software (Cytomation, CO),

Platelet aggregation assay

Platelet aggregation in PRP was measured in a platelet aggregometer (two-sample,

four-channel, model 560 Ca Lumi-aggregometer, Chronolog Corp., Havertown, PAl,

according to the manufacturer's specifications, as a percentage change in light optical

density from baseline (set to 0%) as compared to a reference sample. The maximum

platelet aggregation induced by a potential stimulator was detennined after 6 minutes.

Fresh pPBPC, cryopreserved pPBPC, or cryopreserved baboon PBPC were added to

the baboon PRP in increasing quantities, ranging from lxl05 - Ixl07 cells, and

platelet aggregation was measured. VYhen PBPC were added, an initial increase in

optical density was observed dependent on the final number of particles in solution.

143


Baseline aggregation was then reset at 0%. In other experiments, pPBPC (1xl06 -

lxl07) from growth factor-mobilized and unmobilized miniature swine, or individual

and combined fractions ofpPBPC (2xl05 - 2xl06, as described above), were added to

the PRP. Positive controls of aggregation were baboon PRP to which exogenous

collagen (5 !J.glml) was added. Negative controls consisted of experimental samples of

baboon PRP alone, or to which HBSS was added. Continuous stirring of the sample

with a magnet prevented sedimentation of cells during the assay.

Competitive blocking experiments were perfonned by incubating baboon PRP with an

anti-human P-selectin mAb (R&D Systems) (2.5 - 5.0 f!g/ml), an anti-CDl54 mAb

(100 - 300 f!g/rnl) that would recognize adhesion receptors linked to platelet-cellular

interactions (ref), or eptifibatide (GPIIb/IIIa receptor antagonist) (0.5 - 5.0 ).lg/ml), at

room temperature for 15 minutes. Following this incubation, the extent of baboon

platelet aggregation in the presence of pPBPC was measured.

Immunohistology

The experimental samples, obtained after platelet aggregation assays with baboon or

pPBPC, were centrifuged at room temperature for 10 seconds. The pellet was

resuspended in 20 III supernatant, and snap-frozen in isopentane embedded in TBS

tissue freezing medium (Triangle Biomedical Sciences, Durham, NC). Five J-lm

sections were prepared and stained with standard immunohistochemical staining

techniques using the murine anti-CD62 rnAb (P-selectin, Becton-Dickinson), or the

murine anti-GPIIbIIIa (CD3Ia, R&D Systems, both cross-reactive with baboon), and

porcine pan-leukocyte mAb (l030H-I-19), as well as with a hematoxylin

counterstain.

RESULTS

In vivo experiments

Approximately 70-80% of PBPC remained viable after the freezing/thawing process.

All baboons developed a marked thrombocytopenia «20,000/f!1) almost immediately

144


following transplantation of pPBPC that necessitated platelet transfusions on several

occasions. In baboons that received pPBPC only, platelet recovery was observed

within 5 - 9 days (data not shown), whereas pPBPC transplantation with the NMCR

resulted in sustained thrombocytopenia for 2 - 3 weeks (Fig. I - top left). These

differences were in keeping, at least in part, with the myelosuppression associated

with whole body irradiation. All baboons developed an increase in serum lactate

dehydrogenase level, which was more marked in the baboons receiving pPBPC alone

than in those receiving pPBPC with the NMCR (>!O,OOOU/I and <1,700U/l,

respectively, data not shown). Components of the NMCR (Le. heparin, prostacyclin,

and methylprednisolone) appear responsible for this protective effect (Appel JZ III,

manuscript in preparation). Increases in schistocytes and platelet aggregates on blood

smear were also observed (Fig. 1 - top right). These features were consistent with a

thrombotic microangiopathy. In the three baboons that died, microvascular

thrombosis and interstitial hemorrhage was observed in lungs, kidneys, heart, and

other organs. Immunohistochemical examination revealed deposition of platelets

closely associated with porcine cells (Fig. 1 - bottom).


The addition of baboon PBPC (lxl05 - 2xI0') did not result in measurable

aggregation of baboon platelets as assayed by light aggregometry (Fig. 2 - left).

Increased baseline optical density was, however, observed depending on the number

of baboon PBPC added. The more sensitive immunohistochemical examination of

sedimented samples demonstrated small baboon platelet aggregates in conjilllction

with Ix I 06 baboon PBPC (Fig. 2 - right). Examination of baboon platelets alone, or

platelets plus HBSS (data not shown), did not demonstrate any aggregates. These data

indicate that minimal platelet aggregates are observed in conjunction with baboon

PBPC. These are most likely due to limited interaction between the baboon PBPC

products and platelets under shear stress.

145


_ 1

Figure 1.

, ..

--... -.... t

o .. vs

Top left. Immediate thrombocytopenia following pPBPC infusion in baboons receiving NMCR. The

platelet counts (x 1 x I 0·3/1l1) are plotted against time . Porcine PBPC (total 3x 1 01Q ce lls!kg) were infused

on days 0, I, and 2 (depicted by arrows). Note the rapid fall in platelet count to <100,000/111

immediately after the first pPBPC are infused. The thrombocytopenia is sustained around 10,000 -

20 ,OOOljll for approximately 14 days, in keeping at least in part with bone marrow depression resulting

from whole body irradiation, during which platelet transfusions are required (not shown). Recovery of

platelets to pre-PBPe infusion levels generally took place within 3 - 4 weeks. t signifies the demise of

baboon 869-360 from thrombotic complications.

Top right. Platelet aggregates and schistocytes on blood smear from a representative baboon that

underwent the NMCR and pPBPC transplantation (3xlO ' O !kg). The horizontal arrow indicates baboon

platelet aggregates. The 2 vertica l arrows indicate typical schistocytes.

Bottom. Immunohistochemical examination of frozen lung tissue obtained at autopsy from 869-360 on

day 9 (see Fig. 1 - top left) . The two photomicrographs are serial sections (note the blood vessel in the

lower left comer) at I OOx magnification.

Left. Stained with an anti-CD62 (P-selectin) mAb. Arrow depicts conglomerate of baboon platelets in

microvasculature.

Right. Stained with anti-l 030H 1-19 (pan-pig leukocyte) mAb. Arrow depicts porcine leukocytes in

identical location as baboon platelet aggregate.

146

2 3

J---- \.-1..._--- "

• i' .. !' ,

... --;-~-.. -

Figure 2.

. "

•


,.

~ .' " ,

... ' . • ~ .. •

Left . Graphs depicting the effect of baboon (allo) PBPC on baboon platelet aggregation as measured by

li ght aggregometry. The percentage aggregation is calculated by subtract ing the optical density

observed immediately after the PBPC have been added (set to 0%) from the optical densiry observed

after 6 minutes. Baboon PBPC are added in increasing numbers. The addition of I x I 05 (I), I x I 06 (2),

or 2x I 06 (3) baboon PBPC did not cause macroscopic aggregat ion of baboon platelets.

Right. Immunohi stochemical examination of experimental sample from Fig. 2 - left - 2 ( I x I 06 baboon

PBPe + PRP). Specific staining of platelets was perfonned with an ami-CD62 mAb and leukocytes

were visua lized by hematoxylin counterstain. The horizontal arrow indicates several aggregated

platelets around an activated baboon leukocyte.

2 3

F~ - " , .

!'

, • J

Figure 3.

Left. Graphs depicting the effect of porcine (xeno) PBPC on baboon platelet aggregation as measured

by light aggregometry. Porcine PBPC were added in increasing numbers. The add ition of Ixl05 (I),

lxlO6 (2), or 2xl06 (3) pPBPC induced 17%, 26%, and 35% aggregation of baboon plate lets.

respectively.

Right. Immunohistochemical examination of experimental sample from Fig. 3 - left - 2 ( I x I 06 pPBPC

+ PRP). Specific staining of baboon platelets was perfonned with an anti-CD62 mAb and of porcine

leukocytes with ami- 1030H 1-1 9 mAb. Note the extensive platelet conglomerates incorporating porcine

leukocytes as indi cated by the horizontal arrow.

147


Addition of cryopreserved pPBPC (lxl05 - 2xl06) led to marked aggregation of

baboon platelets that was further determined to be dose-dependent (Fig. 3 - left).

Platelet aggregation approximated 17%,26%, and 35% with addition of lxl05, lxl06,

and 2xl06 pPBPC, respectively. Furthermore, in these samples increased optical

density was observed that was dependent on the number of pPBPC added. Variations

in platelet aggregation could be observed depending On the pPBPC product and the

source of the PRP. In 5 assays, lxl06 pPBPC induced 14% - 63% baboon platelet

aggregation, with a mean of28% (data not ShO\\ll1).

Immunohistochemistry demonstrated mUltiple large platelet aggregates in conjunction

with 1 x 1 06 pPBPC (Fig. 3 - right), when compared to baboon PBPC.

Fresh pPBPC induced more platelet aggregation than cryopreserved PBPC (Fig. 4). In

4 assays, lxl06 fresh pPBPC induced 30 - 79% (mean 48%) platelet aggregation.

Fresh pPBPC contained more granulocytes and platelets than cryopreserved pPBPC,

whereas the latter contained more dead cells (data not shown). The highest percentage

of platelet aggregation (79%) was observed with fresh pPBPC that were obtained

from a pig that had not been mobilized with porcine growth factors. The main

difference found between umnobilized and mobilized pPBPC was the higher

proportion of monocytes in mobilized pPBPC that increased relative to the duration of

growth factor-mobilization (data not shown).

Fresh or cryopreserved porcine granulocytes, CD2+ cells, CDT cells in isolation, or

porcine membranes alone were not able to induce aggregation of baboon platelets

(Table 1). When all subsets of fresh or cryopreserved pPBPC including the

membranes were combined, however, 48% and 14% platelet aggregation was

induced, respectively. The omission of exogenous porcine membranes from the

preparation consistently failed to induce aggregation. Unsorted fresh or cryopreserved

PBPC resulted in 52% and 22% aggregation, respectively.

148


A B

Tim. 1m,",.",

Figure 4.

Graphs comparing the effect of lxlO6 fresh and cryopreserved pPBPC (from the same leukapheresis

product) on platelet aggregation as measured by light aggregometry. Representative experiments are

shovro. Fresh pPBPC induced 32% platelet aggregation, whereas cryopreserved pPBPC induced only

21 % aggregation. Note that the increase in optical density following the addition of frozen/thawed

pPBPC is less than that following the addition of fresh pPBPC, suggesting less exogenous particulate

matter in the cryopreserved product.

Pre-incubation of PRP with anti-P-selectin mAb led to a 60% inhibition of platelet

aggregation when lxl05 cryopreserved pPBPC were added, when compared to

aggregation in the absence of P-selectin (Table 2). Pre-incubation of PRP with anti

CD154 rnAb or eptifibatide resulted in 51% and 93% inhibition of platelet

aggregation, respectively, after 1x106 cryopreserved pPBPC were added (Table 2),

when compared to controls.

149


TABLE 1

BABOON PLATELET AGGREGATION RESPONSES ARE NOT INDUCED BY PURIFIED

FRACTIONS OF PORCINE PBPC

Fractions of pPBPC I Cryopreserved aggregation Fresh aggregation , response (%) response (%)

CD2+ I 2

, CDr 4 I

Granulocytes * 2 0

Exogenous membranes 2 2

Combined fractions ** 48 14 , Combined fractions wlo membranes 4

, 0

Total product pre~cell sorting 52 22

Fractions of pPBPC were obtained by cell sorting (as described).

2xl06 cells/fractions are used in all assays except when otherwise noted.

2xl05 cells were used in this assay as fewer could be obtained.

** Combination of all fractions of pPBPC in equal percentages.

DISCUSSION

Thrombotic microangiopathy in baboons following the transplantation of large

numbers of pPBPC is a serious and potential fatal complication. Furthennore, it also

limits the number of pPBPC that could be transplanted, thereby decreasing the

possibility of induction of mixed hematopoietic chimerism and anticipated

immunologic tolerance. In contrast to the thrombotic microangiopathy following

autologous or allogeneic bone marrow transplantation, the onset of thrombocytopenia

and other thrombotic events was directly related to the infusion of pPBPC to the

baboon, suggesting a direct interaction between pPBPC and baboon platelets. This

was confinned in the baboons that died, where, at autopsy, baboon platelets were

demonstrated in close proximity to porcine leukocytes in the microvasculature of

lungs and kidneys.

150

I


TABLE 2

THE INDUCTION OF BABOON PLATELET AGGREGA nON RESPONSES BY

CRYOPRESERVED PORCINE PBPC IS MODULATED BY ANTI-P-SELECTIN AND ANTl

CDI54 MONOCLONAL ANTIBODIES AND FULLY ABROGATED BY EPTIFIBATIDE

pPBPC Aggregation response (%) Decrease from baseline (%)

Anti-P-selectin mAb * 8 60

Anti-CD154 rnAb ** 22 51

Eptifibatide *** 3 93

lxl06 cells are used in all assays except when otherwise noted.

Baseline aggregation response is obtained in the absence of blocking agents (anti-P-selectin mAb,

anti-CD154 rnAb, or eptifibatide).

Blocking agents were pre-incubated with PRP for 15 minutes at room temperature.

lxlOS cells were used in this assay (for baseline as well as blocking experiment)

as fewer could be obtained. Concentration of anti-P-selectin rnAb was 5.0 ).lg/ml.

** Concentration of anti-CD154 mAb was 300 fig/mi.

*** Concentration of eptifibatide was 5.0 jlg/ml.

In our in vitro studies, porcine PBPC, but not baboon PBPC were able to induce

baboon platelet aggregation in a dose-dependent manner. Immunohistological

examination demonstrated incorporation of porcine leukocytes into baboon platelet

aggregates. Further analysis of the pPBPC product revealed that heightened

aggregation was initiated by fresh rather than frozen pPBPC. Fragmented porcine

erythrocytes, dead cells, and/or platelets also appeared to contribute to this process.

Based on these data, we have hypothesized several mechanisms that may contribute to

this thrombotic microangiopathy. Firstly, dead cells and platelets present in the

pPBPC preparations could provide a greater number of xenogeneic membranes, and

thereby potentiate complement activation (20). Although suppression of complement

activity in baboons undergoing our NMCR is attempted by administering cobra

venom factor, we realize that the complement depletion may be incomplete, and, due

to the mechanism of action of cobra venom factor, residual complement components

151


may be in an activated state. Complement activation has been shovvn to induce and

increase the conversion of prothrombin to thrombin, that can subsequently induce

platelet aggregation (14, 21, 22, 23) and result in the described thrombotic

complications. Secondly, complement has been demonstrated to be associated with

the adhesion of human leukocytes to porcine aortic endothelial cells through P

selectin (24). These interactions may be relevant to om model. As adhesion through

selectin molecules is conserved across mammals (25), porcine leukocytes may

interact with baboon platelets via such adhesion molecules. We have presented further

evidence to this effect by decreasing the induction of pPBPC-mediated baboon

platelet interactions with an anti-P-selectin mAb. Furthermore, since the activation of

complement can lead to upregulation of P-selectin (26) and ~2-integrin (27), porcine

leukocytes may thus adhere to baboon platelets (28, 29). Aggregation of these

activated platelets, initiated by GPlIbIlla cross-linking, could then result. A third

possibility is that porcine monocytes become activated and upregulate adhesion

molecules in the absence of overt complement activation or in the presence of sub

lytic levels of complement. Inflammatory cytokines, such as those released from dead

cells, are associated with an increased expression of adhesion molecules (30). These

cytokines (such as tumor necrosis factor-a) may activate leukocytes, increase the

expression of adhesion molecules (such as P-selectin and ~-integrins) on the cell

surface, consequently activate platelets and induce aggregation responses. These

potential mechanisms and others need to be further elucidated.

We have demonstrated that a thrombotic microangiopathy occurs following the

transplantation of pPBPC in nonhwnan primates, and that this is associated with

porcine leukocyte - baboon platelet interaction. We can now present evidence that

pPBPC can directly induce the aggregation of baboon platelets in vitro. Aggregation

responses can be modulated by anti-CD154 and anti-P-selectin mAbs, and are fully

abrogated by eptifibatide (a potent GPlIbIlla antagonist). The severe thrombotic

microangiopathy that is associated with the transplantation of pPBPC into baboons

may therefore be ameliorated by stringent pmification of the pPBPC product and/or

including eptifibatide to the conditioning regimen. Such an approach may allow for

the transplantation of larger numbers of pPBPC and facilitate the induction of mixed

152


chimerism and immunological tolerance. These studies are currently W1derway in our

laboratory.

REFERENCES

I Cooper DKC, Ye Y, RolfLL Jr, et a1. The pig as a potential organ donor for man. In: Cooper DKC, Kemp L Reemtsma K, et a1. (eds.). Xenotranplantation. Heidelberg, Springer 1991. p48l

2 Sachs DH. The pig as a potential xenograft donor. Vet Imm Immunopath 1994; 43: 185 3 Schmoeckel M, Bhatti FN, Zaidi A, et al. Orthotopic heart transplantation in a transgenic pig-to

primate model. Transplantation 1998; 65: 1570 4 Zaidi A, Schmoeckel M, Bhatti F, et al. Life-supporting pig-to-primate renal xenotransplantation

using genetically modified donors. Transplantation 1998; 65: 1584 5 Sablinski T, Emery DW, Monroy R, et aL Long-term discordant xenogeneic (porcine-to-primate)

bone marrow engraftment in a monkey treated with porcine-specific growth factors. Transplantation 1999; 67: 972

6 Tomita Y, Yoshikawa M, Zhang QW, et a1. Induction of permanent mixed chimerism and skin allograft tolerance across fully MHC-mismatched barriers by the additional myelosuppressive treatments in mice primed with allogeneic spleen cells followed by cyclophosphamide. J Immunol

2000; 165: 34 7 Wekerle T, Kurtz J, Ito H, et a1. Allogeneic bone marrow transplantation with co-stimulatory

blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat Med 2000;6:464

8 Huang CA, Fuchimoto Y, Scheier-Oolberg R, et a1. Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model. J Clin Invest 2000; 105: 173

9 Kawai T, Ponce let A, Sachs DH, et a1. Long-term outcome and al1oantibody production in a nonmyeloablative regimen for induction of renal allograft tolerance. Transplantation 1999; 68: 1767

10 Alwayn IPJ, Buhler L, Basker M, et al. Coagulation! Thrombotic disorders associated with organ and cell xenotransplantation. Transpl Proc 2000; 32: J 099.

11 Sablinski T, Emery DW, Monroy R, et a1. Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors. Transplantation 1999:67:972

12 Buhler L, Basker M, Alwayn IPJ, et al. Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Transplantation 2000; 70: 1323

13 Holler E, Ko!b HJ, Hiller E, et al. Microangiopathy in patients on cyc\osporine prophylaxis who developed acute graft-versus-host disease after HLA-identica! bone marrow transplantation. Blood 1989: 73: 2018

14 Robson SC, Schulte am Esch J 2nd, Bach FH. Factors in xenograft rejection. Ann NY Acad Sci 1999; 875: 261

15 Kozlowski T, Shimuzu A, Lambrigts 0, et al. Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption. Transplantation 1999; 67: 18

16 Buhler L, Awwad M, Basker M, et al. High-dose porcine hematopoietic cell transplantation combined with C040 ligand blockade in baboons prevents an induced anti-pig humoral response. Transplantation 2000; 69: 2296

17 Watts A, Foley A, Awwad M, et al. Plasma perfusion by apheresis through a Gal immunoaffinity column successfully depletes anti-Gal antibody - experience with 320 aphereses in baboons. Xenotransplantation 2000; 7: 181

18 Kozlowski T, Monroy R, Xu Y, et a1. Anti-aGal antibody response to porcine bone marrow in unmodified baboons and baboons conditioned for tolerance induction. Transplantation 1998; 66: 176

19 Nash K, Chang Q, Watts A, et al. Peripheral blood progenitor cell mobilization and leukapheresis in pigs. Lab Anim Sci 1999; 49: 645

20 Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997; 89: 1121

153


21 Robinson RA, Worfolk L, Tracy PB. Endotoxin enhances the expression of monocyte prothrombinase activity. Blood 1992; 79: 406

22 Minnema MC, Pajkrt D, Wuillemin WA, et al. Activation of clotting factor XI without detectable contact activation in experimental human endotoxemia. Blood 1998; 92: 3294

23 Peerschke EI, Jesty J, Reid KB, et al. The soluble recombinant fonn ofa binding protein/receptor for the globular domain ofClq (gClqR) enhances blood coagulation. Blood Coagul Fibrinolysis 1998;9:29

24 Rollins SA, Johnson KK, Li L, et al. Role of porcine P-selectin in complement-dependent adhesion of human leukocytes to porcine endothelial cells. Transplantation 2000; 69: 1659

25 Jalkanen S, Reichert RA, Gallatin WM, et al. Homing receptors and the control oflymphocyte migration. lmmunol Rev 1986; 91: 39

26 Mulligan MS, Schmid E, Till GO, et al. C5a-dependent up-regulation in vivo oflung vascular Pselectin. J Immunol 1997; 158: 1857

27 Jagels MA, Daffern PJ, Hugli TE. C3a and C5a enhance granulocyte adhesion to endothelial and epithelial cell mono layers: epithelial and endothelial priming is required for C3a-induced eosinophil adhesion. Immunophannacology 2000; 46: 209

28 Brown KK, Henson PM, Maclouf J, et al. Neutrophil-platelet adhesions: relative roles of platelet Pselectin and neutrophil ~2 (CDI8) integrins. Am J Resp Cell Mol BioI 1998; 18: 100

29 Diacovo TG, Roth SJ, Buccola JM, et aJ. Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action ofP-selectin and the beta 2-integrin CD1Ib/CDI8. Blood 1996; 88: 146

30 Teplyakoy AI, Pryschepova EY, Kruchinsky NG, et al. Cytokines and soluble cell adhesion molecules. Possible markers of inflammatory response in atherosclerosis Ann NY Acad Sci 2000; 902: 320

154

7 Immunosuppressive therapies and platelet aggregation in haboons

Adaptedfrom: Appel J2 IIr', Alwayn IPj', Buhler L, DeAngelis HA, Cooper DKC·, and Robson SC·, Modulation of platelet aggregation in baboons: implications for mixed chimerism in xenotransplantation. 1. The roles of individual components of a transplantation conditioning regimen and of pig peripheral blood progenitor cells. Transplantation 2001. In press. & Appel JZ III, Alwayn IPl, Buhler L, Correa LE, Cooper DKC·, and Robson SC·. Modulation of platelet aggregation in baboons: implications for mixed chimerism in xenotranspiantation. 2. The effects of cyclophosphamide on pig peripheral blood progenitor cell-induced aggregation. Transplantation 2001. In press . • Authors contributed equally • Joint senior authors.

PUBMED-ID

HYPERLINK "/pubmed/11602859"Modulation of platelet aggregation in baboons: implications for mixed chimerism in xenotransplantation. I. The roles of individual components of a transplantation conditioning regimen and of pig peripheral blood progenitor cells. Appel JZ 3rd, Alwayn IP, Buhler L, DeAngelis HA, Robson SC, Cooper DK. Transplantation. 2001 Oct 15;72(7):1299-305. PMID: 11602859 [PubMed - indexed for MEDLINE]

PUBMED-ID

HYPERLINK "/pubmed/11602860"Modulation of platelet aggregation in baboons: implications for mixed chimerism in xenotransplantation. II. The effects of cyclophosphamide on pig peripheral blood progenitor cell-induced aggregation. Appel JZ 3rd, Alwayn IP, Correa LE, Cooper DK, Robson SC. Transplantation. 2001 Oct 15;72(7):1306-10. PMID: 11602860 [PubMed - indexed for MEDLINE]


ABSTRACT

Introduction. The induction of tolerance to pig antigens in primates should facilitate

the development of successful clinical xenotransplantation protocols. The infusion of

mobilized porcine peripheral blood leukocytes (PBPC) into splenectomized pre

conditioned baboons, intended to induce mixed hematopoietic cell chimerism, however,

results in thrombocytopenia and a severe thrombotic microangiopathy (TM). As the

mechanisms responsible for this phenomenon are unclear, we have explored the effects

of individual components of the conditioning regimen, of therapeutic adjuncts and of

PBPCs on platelet aggregation.

Methods. Groups of splenectomized baboons (n=2 in each group) were treated with

single components of the conditioning regimen - whole body irradiation (\VBI), anti

thymocyte globulin (ATG), extracorporeal irnmunoadsorption (EIA), mycophenolate

mofetil (MMF), anti-CD40L mAb, cobra venom factor (CVF), pig hematopoietic

growth factors (interleukin-3 (pIL3) and stem cell factor (PSCF» - or with potential

adjuncts, cyclophosphamide (CPP), prostacyclin (PGI2), heparin, methylprednisolone,

and eptifibatide. Blood samples were collected and platelet-rich plasma (PRP) was

prepared. Using light transmission aggregometry, the extent of aggregation induced by

platelet agonists (Thrombin, adenosine diphosphate (ADP), collagen, ristocetin, and

arachidonic acid) was detennined in vitro. PRP was also prepared from untreated

baboons and baboons receiving either eptifibatide (a GPIIb/IIIa antagonist) or CPP and

PBPC were added and platelet aggregation was measured in the absence of exogenous

platelet agonists.

Results, WEI, ATG, MMF, anti-CD40L mAb, CVF, pIL3 + pSCF, and PGI2 had no

effect on purified baboon platelet aggregation profiles in vitro. CPP or eptifibatide

markedly inhibited platelet aggregation induced by all standard agonists. Thrombin

induced platelet aggregation was inhibited by EIA or heparin, and ADP-induced

aggregation was inhibited to some extent by methylprednisolone. In vitro addition of

PBPC to PRP stimulated platelet aggregation in the absence of any agonists. Prior

treatment of baboons with eptifibatide or CPP, however, inhibited this effect by 70-80%

156

Immunosuppressive therapies and platelet aggregation

and 55%, respectively. TM was not evident in baboons receiving a conditioning

regimen that included CPP instead of WBI.

Conclusions. Aggregation of baboon platelets and TM is directly induced by PBPC.

but not by individual components of the conditioning regimen. CPP has direct anti

aggregatory properties and may provide an alternative strategy to WBI. GPilb/IIla

antagonists, such as eptifibatide, interfere directly with xenogeneic PBPC-platelet

interactions and may further ameliorate TM in the pig-to-primate modeL

INTRODUCTION

Because of the limited availability of suitable human organ donors,

xenotransplantation has received considerable attention in recent years. For several

reasons, the pig is considered the best candidate for this purpose (1-3). However, the

transplantation of porcine tissue into humans or non-human primates elicits a severe

and rapid rejection response, resulting in graft loss, even when intensive

immunosuppressive therapy is administered (4).

One approach that would overcome the pig-to-primate xenogeneic barrier is the

development of donor-specific unresponsiveness, or transplantation tolerance (5).

Tolerance has been induced in several allogeneic models using a variety of

approaches. Of these, perhaps the most successful protocols are those that establish

mixed hematopoietic cell chimerism by infusing donor bone marrow or hematopoietic

progenitor cells into conditioned recipients (6).

Our center has infused porcine hematopoietic progenitor cells into conditioned

primates in an attempt to induce a state of pig-primate mixed chimerism to promote

xenogeneic tolerance (5). Leukocytes are mobilized from porcine bone marrow ill

vivo using pig-specific hematopoietic groVvih factors (7-9). These mobilized

peripheral blood leukocytes (PBPC), that contain approximately 2% hematopoietic

progenitor cells, are collected by leukapheresis (9). The PBPC are then infused into

baboons that have undergone a conditioning regimen modified from those that have

met with success in allogeneic models (See Chapter I. Figure 3). This preparative

157


regimen entails thymic irradiation and non-lethal myelosuppression in the form of

whole body irradiation (WEI). Baboons receive anti-thymocyte globulin (ATG) and

undergo a process of extracorporeal immunoadsorption (EIA) of anti-Gal (al-3) Gal

(Gal) natural antibodies. Baboons also receive immunosuppression, in the fonn of

mycophenolate mofetil (MMF) and anti-CD40L mAb, cobra venom factor (CVF), and

pig-specific hematopoietic growth factors, pig interleukin-3 (pIL3) and pig stem cell

factor (PSCF) (10). Once this regimen has been completed, large numbers of porcine

PBPC (2-4 x 101O/kg) are infused intravenously.

Although transient engraftment of pig hematopoietic progenitor cells (detectable in

the blood by pol)'lllerase chain reaction and by flow-activated cell sorting) has been

achieved in two of eight baboons receiving this regimen, a severe thrombotic

microangiopathy (TM) inevitably develops shortly after the infusion of PBPC (7).

This is manifest by pronounced thrombocytopenia (platelet count < 20,000) (See

Chapter 5, Figure 2), schistocytosis, and elevation of lactate dehydrogenase. In many

regards, this syndrome resembles the TM observed in approximately 14% of bone

marrow transplant recipients (11) but, in the xenogeneic combination, develops much

earlier (i.e. long before, not after, engraftment). Prothrombin time (PT), partial

thromboplastin time (PTT), and fibrinogen levels, although transiently disturbed by

EIA, typically remain unaffected. The condition can be fatal in experimental animals,

and autopsies reveal microvascular thrombosis and vascular injury in several major

organs, including the heart, lungs, and kidneys (Figure I) (7).

TM represents a considerable barrier to the achievement of xenotransplantation

tolerance in this model because it may be associated with significant morbidity and it

limits the total number of PBPC that can be infused, therefore reducing the likelihood

of establishing mixed hematopoietic cell chimerism. Thus, an improved

understanding of the mechanisms underlying the platelet activatioillaggregation

observed in baboons receiving \VBI and PBPC may suggest ways of preventing these

sequelae, thereby facilitating mixed chimerism and the induction of

xenotransplantation tolerance.

158


• Figure I.

Baboons receiving WBI develop thrombotic microangiopathy after infusion of pig PBPC.

Microvascular thrombi with platelet sequestration develop in organs such as the kidney (stained for P

selectin).

Cyclophosphamide (CPP) has been used as an alternative to irradiation in some

instances prior to clinical bone marrow transplantaion (12). A prodrug, CPP is

biotransformed by a microsomal oxidase system in the liver to active metabolites.

Some of these species inhibit the growth of rapidly proliferat ing celis, most likely by

cross-linking DNA, thereby exerting an immunosuppressive and, possibly,

myelosuppressive effect. Additionally, the administration of CPP to some patients

with TM of various etiologies has proved therapeutic (13-16). Thus, CPP may be a

favorable alternative to WBI in this pig-to-primate model of mixed chimerism and

tolerance induction.

In this study, standard light transmission aggregometry was utilized to assess the

effects of the individual components of this complex conditioning regimen and of

PBPC on platelet aggregation in vitro. Furthermore, the anti-thrombotic properties of

c pp and of several potential adjuncts to the current conditioning regimen, including

159


PGI2, heparin, methylprednisolone, and eptifibatide (a GPIIb/IIIa platelet receptor

antagonist) were examined.

MATERIAL & METHODS

Animals

Baboons (Papio anubis, n=12, Biomedical Resources Foundation, Houston, TX),

weighing 9-13 kg, were used to test experimental agents in vivo and as a source of

platelets for in vitro experiments. All animals had been previously splenectomized

through a midline laparotomy. Normal platelet counts and coagulation parameters

were present in all baboons at the onset of the present study. All experiments were

performed in accordance with the Guide for the Care and Use of Laboratory Animals

prepared by the National Academy of Sciences and published by the National



Surgical Cannulation of Vessels

Baboons were anesthetized and the carotid artery and internal jugular vein were

cannulated with silastic catheters as described previously (17). A jacket and tethering

system was utilized, which allowed continuous or intermittent administration of

intravenous (IV) fluids and/or drugs, withdrawal of blood, monitoring of arterial

blood pressure, and sedation (as needed) for the baboon. Cefazolin sodium (500 mg

IV) was given daily as prophylaxis against infection.

Individual Components of the Conditioning Regimen

All immunomodulatory procedures, agents, or combinations of agents, were tested in

vivo in at least two baboons (Table 1). Serial blood samples were taken in order to

monitor platelet count, PT, PTT, fibrinogen, fibrinogen degradation products, white

blood cell counts, and hematocrit (Hematology Laboratory, Massachusetts General

Hospital). Seven baboons were used to test more than one agent, but platelet

aggregation and coagulation parameters were allowed to return to baseline before

160


subsequent drugs were administered. Most agents were infused continuously

although bolus injections were used in some instances, as indicated in Table 1.

Collection of Mobilized Peripheral Blood Leukocytes (PBPC)

PBPC were collected from miniature swine using a 16-day leukapheresis protocol

described previously (9). Pigs were treated with recombinant hematopoietic growth

factors (8-10) before and during the period of leukapheresis. On days 0-15, pIU and

pSCF were administered daily by subcutaneous injection, each at a dose of 100

r;g/kg/d. Human granulocyte colony stimulating factor (Arngen, Thousand Oaks, CAl

was administered subcutaneously at 10 J..Lglkgld on the evening before each

leukapheresis procedure. Leukapheresis was performed on days 5-9 and 12-16 using

a Cobe Spectra apheresis machine (Cobe, Lakewood, CO). This leukapheresis

product was washed and, following lysis of red blood cells, the remaining PBPC were

frozen in 5% dimethylsulfoxide.

Platelet Aggregation Assay

At intervals before, during and after the administration of each agent, or before and

after a procedure (e.g. EIA, WBI), 10 ml citrated blood was drawn from the baboon

and centrifuged at 280 g for 15 minutes at 22°C. The supernatant, consisting of

platelet-rich plasma (PRP), was removed and the remaining pellet was centrifuged at

2,400 g for 2 minutes at 22 'C to obtain platelet-poor plasma. The final PRP was

transferred in 400 IJ.I aliquots to siliconized glass cuvettes, comprising the

experimental samples. A 400-)11 aliquot of platelet-poor plasma was used as a

reference sample, to which platelet aggregation in PRP samples could be compared.

161


TABLE I

AGENTS I PROCEDURES ADMINISTERED TO BABOONS

AgentJProcedure Dose Administered References Suggesting

Tested Pro~/Anti~ Thrombotic i i Properties

! Whole body irradiation i 150 cGy/d x 2d 18-21

Anti~thymocyte globulin 50 mg/kg/d elv x 3d 22

: Extracorporeallmmunoadsorption i Daily x 3d 23,24

i. Mycophenolate mofetil llO mg/kgld CIY 22,25

Anti-CD40L mAb 40 mg/kg xl I 26-29

i Cobra venom factor I 100-200 Ulkgld x 3d I 30-32

: :ecombinant pIL3 200 !J.g/kg bolus, I 400 ~glkg/d CIY x 2d

, , I Recombinant pSCF

, 1 mg/kg bolus,

2 mg/kg/d elv x 2d

Cyclophosphamide I 40 mg/kg/d x 3d 12-16,33,34

Prostacyclin (PGI2) 20-30 ng/kg/min CIV x 3d 35-37

Heparin sodium 10 50 Vlkg/hr CIV x 5d 38,39

Methylprednisolone 2 mg/kg twice daily x 3d 40

Eptifibatide (GPllb/Ula 180 ).Lg/kg bolus, 41,42

! antagonist) 2.0-7.5 ).Lglkg/min CIV x Id

pIL3 = porcine interleukin 3, pSCF = porcine stem cell factor, CIY = continuous intravenous infusion

Four of five standard platelet agonists (thrombin (1.25 Ulml), adenosine diphosphate

(ADP) (20 ).LM), collagen (10 ).Lg/ml), and ristocetin (1.25 ).Lg/ml)) were added to the

experimental samples, and the extent of aggregation over a period of six minutes was

measured using a platelet aggregometer (two-sample, four-charmel, model 560 Ca

Lumi-aggregometer, Chronolog Corp., Havertown, PA). Because ristocetin (which

acts via GPIb receptors in association with v\VF) was found to be a less potent

agonist, arachidonic acid (which is converted into pro-aggregatory metabolites PGH2

162

I I i

i

i

I


and TXA2) (1.2 mM) was used in some instances. By adding a small rotating magnet

to each sample, shear stress was created in order to simulate intravascular flow

conditions.

VVhenever PBPC were to be added to baboon platelets in vitro, blood was drawn from

untreated baboons and, in the manner described above, PRP was prepared.

Immediately prior to use, PBPC were rapidly thawed in a water bath at 37°C and

were washed twice with calcium-, magnesium-, and phenol red-free Hanks balanced

salt solution. In the absence of any agonists, increasing numbers of PBPC (1 x 105, 2

x 105, 1 x 106

, and 2 x 106) were added to separate PRP samples and aggregation was

measured.

Immunohistochemical Staining of PBPC-stimulated Aggregates

Experimental PRP samples to which PBPC had been added were centrifuged at

10,000 rpm for 20 seconds. The supernatant was aspirated from these and the

remaining pellet was resuspended in 20 /.11 supernatant, and frozen in TBS tissue

freezing medium (American Mater-Tech Scientific, Lodi, CA). From these frozen

samples, 5 )..tm-thick sections were prepared and subsequently stained with anti-P

selectin or 1 030H-l-19, a pan-pig monoclonal antibody developed by our laboratory.

RESULTS

Effects of Individual Components of the Conditioning Regimen on Platelet

Aggregation

Table 2 summarizes platelet aggregation (relative to baseline) measured in PRP

prepared from baboons treated with each of the individual components of the

conditioning regimen. A change in platelet aggregation was considered to be

substantial if it differed by more than 20% from baseline measurements and occurred

in both baboons tested.

Exposure to \VBI did not affect platelet aggregation, which was assessed 48 hours

following the second fractionated dose. Following WEI, platelet counts in both

163


baboons decreased gradually, reaching values approximating 20% of baseline

measurements after 8 and 10 days, respectively. Coagulation parameters and

hematocrit remained stable during this period. Administration of CPP on three

consecutive days (a total dose of 120 mg/kg), however, resulted in a progressive

inhibition of agonist-stimulated platelet aggregation that was most pronounced after

approximately 6-8 days (Figure 2). Aggregation induced by thrombin, ADP, collagen,

and arachidonic acid was decreased from baseline by as much as 62%, 85%, 84%, and

95% respectively, in one baboon and by 74%, 98%, 83%, and 98% in another.

Fourteen days after administration of the drug, platelet aggregation returned to pre

treatment levels in both baboons. Coagulation parameters were not altered

significantly during the experiment. CPP-treated baboons experienced a progressive,

but transient, decrease of white blood cells (from approximately 9,000/f'l to 900/f'I

and from 12,000/f'l to 800/f'I over 7 days) and of platelets (from approximately

310,000/f'l to 66,000/1-'1 and from 300,000/1-'1 to 170,000/f'l over 11 days), which

returned to pre-treatment values 16 days following administration of the drug.

To verify that the reduction in platelet aggregation was independent of platelet count,

PRP was prepared from untreated baboons. Baseline platelet aggregation induced by

thrombin, ADP, collagen, and arachidonic acid was 27%, 30%, 62%, and 20%,

respectively. When diluted (from an initial count of 540,000 platelets/f'l) as much as

eight-fold with calcium-, magnesium-, and phenol red-free Hanks balanced salt

solution, platelet aggregation induced by thrombin, ADP, and collagen was unaffected

(22%, 33%, and 65%, respectively). Similar results were obtained when PRP was

diluted with platelet-poor plasma.

164

100

80

c: o 60 ~ Cl i'! Cl 40 :f Q;

~ 20

0::

o

Figure 2.

, •

I e-

Thrombin AOP


'n n · i

If n J I

Collagen Arachidonic Acid

Agonist

o Baseline

OCPP 04

OCPP 07

IOCpp 09

OCPP0141

Effect of the in vivo administration of cyclophosphamide (total dose 120 mgikg) on in vitro baboon

platelet aggregation. Platelet aggregation by all agonists was partially inhibited 6-! I days after

cyclophosphamide administration. ADP = adenosine diphosphate.

The process of EIA had a transient inhibitory effect on thrombin-induced platelet

aggregation (by 98%) (Table 2). Coagulation parameters. measured at the same time,

were prolonged (PT >30 s, PTT > 80 s) but recovered over a period of 12-18 hours.

Administration of ATG, MMF, anti-CD40L mAb, or CVF to individual baboons,

however, had no substantial effects on the aggregation of baboon platelets. As

anticipated, baboons receiving ATG developed leukopenia, but no thrombocytopenia,

and this was rapidly reversed upon discontinuation of treatment. Laboratory

parameters were otherwise unchanged in these baboons.

Inconsistent effects on platelet aggregation were observed in baboons receiving either

hematopoietic growth factors (pIL3 + pSCFl or PGI2. Ristocetin-induced platelet

aggregation was slightly increased (by 25%) in one baboon receiving pIL3 and pSCF.

Platelet aggregation was unaffected in baboons receiving PGI2 20 ng/kg/min,

165


although ADP- and collagen-induced platelet aggregation was inhibited by 24% and

23%, respectively, in one baboon receiving 30 nglkglmin. No disturbances of vital

signs, coagulation parameters, or other adverse effects were noted in these baboons.

As predicted, heparin reduced thrombin-induced platelet aggregation In a dose

dependent manner. Little effect on global platelet parameters and aggregation

responses was apparent in baboons maintained on a continuous infusion of 10

Ulkg/hour. However, considerable inhibition of thrombin-induced aggregation (by

90%) was evident at 50 Ulkglhr. PTT was prolonged to > 150 seconds in contrast to

baseline parameters of < 30 seconds.

In both baboons that received methylprednisolone, platelet aggregation induced by

ADP was inhibited (by 43% and by 50%). Arachidonic acid-induced platelet

aggregation was inhibited in one baboon by up to 93% on two separate days, but was

unaffected in a second baboon.

The administration of eptifibatide resulted in potent and global inhibition of platelet

aggregation (Figure 3). This was dose-dependent, such that continuous infusion at 2.0

)lglkglmin had little effect (0-48% inhibition), whereas 7.5 )lglkglmin inhibited

aggregation induced by all agonists tested (by 84 - 99%). The onset of this effect was

rapid, evident as early as 30 minutes after initiation of the drug infusion and was

rapidly reversible, platelet aggregation returning to baseline levels within two hours

after discontinuation of therapy.

166

100 c

c: 2 60

'" Q)

1!

.

81 40 • .0: 1ii

~ 20 0:

o

Figure 3.

n ! .

1

Thrombin ADP


I

"I 0 Baseline--

o Eptifibatide IV 1- , 2.0 ~g/kg/min

; 0 Eptifibatide IV , 5.0 Ilg/kg/min

o Eptifibatide IV

: 7.5 )lg/kg/min )1 DAfter Drug

;j Cessation (2hrs.)

.

Collagen Arachidonic

Acid

Agonist

Effect of the in vivo administration of eptifibatide on in vitro baboon platelet aggregation. Eptifibatide

inhibited platelet aggregation induced by all agonists, and had an increasing inhibitory effect at higher

dosages. ADP = adenosine diphosphate.

Vital signs, coagulation, and other laboratory parameters were unaffected at all doses

tested, and bleeding episodes or other adverse effects were not observed.

Effect of PBPC on Platelet Aggregation

In vitro, the addition of porcine PBPC to PRP from untreated baboons stimulated

platelet aggregation in the absence of any agonist. and increased with the number of

PBPC added (Figure 4A & B). Although no aggregation was detected when I x 105

cells were added, the addition of 2 x 105, I X 106

, and 2 x 106 PBPC induced

aggregation by as much as 17%, 26%, and 35%, respectively, in one baboon. This

trend was apparent in a total of four experiments using PBPC and PRP from different

pigs and baboons. Immunocytochemistry using anti-P-selectin and/or the pan-pig

preparation, 1030H-I-19, demonstrated the aggregation of baboon platelets around

porcine leukocytes (See Chapter 6, Figure 3). When PBPC were added in vitro to

167


PRP prepared from baboons treated with eptifibatide (7.5 ).lg/kg/min IV), platelet

aggregation was prevented by > 70% (Figure 4C). Similarly, PBPC-induced platelet

aggregation was inhibited by 55% in PRP prepared from a baboon 6 days after the

administration ofCPP (120 mg/kg total dose) (Figure 4D). In baboons treated with a

CPP-based conditioning regimen instead of \VBI, TM was not evident following the

infusion of PBPC. Although platelet counts in CPP-treated baboons decreased to

some extent, the profound thrombocytopenia and thrombotic complications

characteristic of VYBI-treated baboons did not develop and platelet counts recovered

earlier (Figure 5 - Top). Schistocytosis was not detected and lactate dehydrogenase

levels were unaffected in these CPP-treated baboons (Figure 5 - Bottom).

Figure 4.

Z' .~

~ ~

(B) 22 %

~~_gregation

:: I(C) ~' i~---- ;=="-::-"C:"--' g ~ 5%

Aggregation

(D)

I.J~ I

;-1 ---=, 0-:-:-% ---,

. . Aggregation

--- Time ---4

Platelet aggregation curves of nai've baboons obtained after in vitro addition of porcine PBPC.

(A) Addition of Ix105 porcine PBPC to baboon PRP containing approxiamte[y lxl08 platelets led to

11% aggregation. (8) Addition of 2 x 106 PBPC resulted in 22% platelet aggregation. (C) When

platelets were prepared from the same baboon after treatment with eptifibatide, platelet aggregation

induced by 2 x 106 PBPC was inhibited by > 70% (to 5%). CD) Platelet aggregation induced by 2 x 106

PBPC was inhibited by 55% (to 10%) in PRP prepared from the same baboon 6 days after treatment

with C?P.

168


TABLE 2

EFFECT OF VARIOUS IN VIVO AGENTS/PROCEDURES ON IN VITRO BABOON

PLATELET AGGREGATION

Platelet Agonist

Agent/Procedure c c c :c • ~ 5 .. " Tested N Q ~ v 0 E - ~ "0 .c U ~ ...

i

WBI 2 - - - -

Anti·thymocyte globulin 2 - - -It -, i EIA 2 ... - - -

Mycophenolate mofetil 2 - -It - -Anti·CD40L mAb 2 - - i - -Cobra venom factor 2 - -

i -It -

pIL3 + Pscf 2 - - I - -It

Cyclophosphamide 2 ... ... I

... NA

PGI2 (20 ng/kg/min) 2 - - - NA

PGI2 (30 nglkglm;n) 2 - -It I -It NA

Heparin 2 ... - I - NA

Methylprednisolone 2 - ... i - NA

Eptifibatide 2 + + + NA

,} or t indicates a change in platelet aggregation >20% from baseline.

- indicates a change of <20%. NA indicates agonist not used in corresponding experiment.

WBI = whole body irradiation, EIA = extracorporeal immunoadsorption, pIU = porcine

interleukin 3, pSCF = porcine stem cell factor, PGI2 = prostacyclin, ADP = adenosine

diphosphate.

:s v ~ v

'= 0 :s .c v • -~

NA

NA

NA

NA

NA

NA

NA

... -

--

-It

+

169

I


700. ~Cyclophosphamide ~-WBI

600~

500'

400

300

200

100~

O-------c----c~~~~~~~~--~~~cc -10 -5 0 5 10 15 20 25

WBI 111 DAYS or mPL

Cyclophosphamide

w 2000- ~Cyclophosphamide i --WBi ~

Z W (!) o 0::

1600j

o ::i' 1200 >--:r:::> W::::. Cl W f-

~ () « -'

Figure 5.

800

400l

O~i---c--~----~--~~~~~~~ -10 -5 2115 10 15 20 25

WBI I DAYS or mPL

Cyclophosphamide

Platelet count and lactate dehydrogenase levels in baboon recipients of PBPC treated with WBI- or

cyclophosp:1amide-based conditioning regimens. (Top) Thrombocytopenia is much less pronounced

and (Bottom) lactate dehydrogenase levels are more stable in baboons receiving cyclophosphamide

than in those undergoing WBf.

170


DISCUSSION

Substantial advances in xenotransplantation have enabled hyperacute rejection to be

overcome (4). The onset of acute vascular rejection to be delayed in part by the

administration of intensive immunosuppressive therapy. However, even if acute

vascular rejection (delayed xenograft rejection) can be prevented, the acute cellular

response is likely to be strong. Long-term survival of discordant xenografts may

therefore depend on the successful induction of transplantation tolerance.

By infusing donor hematopoietic progenitor cells into recipients this way, and

inducing a state of mixed hematopoietic cell chimerism, tolerance has been achieved

in allogeneic animal models (6) and, recently, in a human renal allograft recipient

(43). Using a complex conditioning regimen (as described above) (4, 7), transient

bone marrow engraftment of pig progenitor cells has been achieved in some baboons

pretreated with \VBI and thymic irradiation. However, baboons undergoing VVBI

exhibit a severe TM immediately after the infusion of PBPC (7).

The current study investigated the roles of the individual components of the

conditioning regimen, and of PBPC, in the aggregation of baboon platelets.

Furthermore, it assessed the anti-thrombotic properties ofCPP, an alternative to \VBI,

and of several potential adjuvants, including PGI2, heparin, methylprednisolone, and

eptifibatide, a GPIIb/IHa platelet receptor antagonist.

\\!hen administered individually to baboons, no single component of the VVBI-based

conditioning regimen increased platelet aggregation. Porcine PBPC, when added to

baboon platelets in vitro, initiated aggregation in the absence of any platelet agonists.

Immunocytochemistry, using anti-human P-selectin and 1030H-1-19 (a pig-specific

stain), confirmed the formation of PBPC-platelet aggregates. This phenomen

increased with larger numbers of PBPC and suggests that they playa primary role in

the fonnation of platelet microthrombi in vivo. It is likely that the aggregation of

platelets and the sequestration in the microvasculature contribute to the

thrombocytopenia and TM observed in conditioned baboons after PBPC infusion.

171


The observation that \VBI had no effect on platelet aggregation is of interest as the

formation of platelet microthrombi can occur following irradiation in vivo (20, 21).

The detrimental effects of irradiation on the vascular endothelium are well

documented (18, 19) and include endothelial cell activation (indicated by upregulation

of ICAM-l and P-selectin), vacuolization, and separation from the basement

membrane (20). As WBI does not affect the inherent tendency of platelets to

aggregate, as indicated by the results of this study, the formation of microthrombi in

irradiated patients may depend largely on endothelial damage. In contrast to the TM

observed in baboon recipients of porcine PBPC, the clinical sequelae of bone marrow

allotransplantation typically require several days as opposed to hours to become

manifest (11). Although irradiation may inflict endothelial damage in the baboons in

our studies, which may lead to the formation of rnicrothrombi in some instances, it is

unlikely that this contributes primarily to the TM observed immediately following the

administration of PBPC.

Vlhen administered to baboons, CPP inhibited platelet aggregation induced by all

agonists. This effect may explain why treatment with CPP instead of \VBI prior to the

infusion of PBPC protected baboons against TM. In contrast to \VB1-treated baboons,

those receiving CPP appeared to tolerate the infusion of PBPC without major

complications and with only a minor reduction in platelet count (Figure 5 - Top).

However, engraftment may be compromised as it was not observed in baboons

receiving CPP. CPP may be a less effective mode of myelosuppression in this model,

or it may not create the bone marrow "space" necessary for PBPC engraftment.

However, the use of CPP as an alternative, or in combination with a lower dose of

\VBl, may yield certain advantages.

Although not reported here, the infusion of PBPC into baboons in the absence of the

conditioning regimen initiated a state nearly identical to the TM seen in baboons

receiving the full regimen, confirming the primary role of PBPC in this syndrome

(See Section 4, Chapters 5 & 6). This finding also supports the conclusion that CPP is

protective against TM, since this syndrome develops in baboons (with or without

172


\VBl), but is not evident in those receiving CPP. This also demonstrates that \VBI is

not necessary for the initiation of TM, although it may exacerbate this state by

delaying marrow regeneration. Moreover, additional effects of WBI, such as

endothelial cell damage, may also contribute to the formation of microthrombi.

The inhibition of platelet aggregation by CPP was not apparent until approximately

one week after infusion of the drug. Thus, the direct actions of one or several

metabolites of CPP on platelets could contribute. Two of these, 4-hydroxy-CPP and

acrolein, have been implicated in at least one other report (17). Data from preliminary

experiments in our laboratory suggest that the effects of CPP on baboon platelets

result from the modification of signal transduction pathways that are involved in

normal aggregation (data not shown).

Because the cytotoxic effect of CPP metabolites has been attributed to their ability to

cross-link DNA nuc1eotides, our laboratory has hypothesized that such complexes

could bind purinergic receptors resulting in blockade at the platelet surface. However,

using serotonin and epinepherine, which would bypass purinergic receptor blockade,

indicate that this is not the case (data not sho\VIl).

The maximal CPP-induced inhibition of platelet aggregation was apparent on days

when platelet counts approached their nadirs (66,OOO/f'1 and 170,OOO/f'I, respectively).

Previous reports have indicated that ADP- or collagen-induced platelet aggregation is

sustained at platelet counts as low as 50,OOO/f'1 (44). Our control studies, in which

PRP (with an initial platelet count of 540,OOO/f'I) was diluted as much as eight- to

twelve-fold, confirmed these findings for ADP and collagen, as well as for thrombin.

Thus, a low platelet count alone would not account for this decrease in platelet

aggregation. It is possible that this decrease in platelet count reflects CPP-induced

megakaryocyte depletion or inhibition, resulting in temporarily diminished platelet

regeneration. Subsequent recovery of megakaryocyte function and production of new

platelets would account for the concomitant increase in platelet count and return of

platelet aggregation to baseline.

173

Section IV ~ Chapter 7

The EIA of anti-Gal antibodies transiently prevented thrombin-induced platelet

aggregation. The activation of platelets and coagulation factors by circulation through

the irnmunoadsorption apparatus could contribute to this effect. This may have been

exacerbated further, however, by the high concentration of citrate used as an

anticoagulant during EIA, which may have had marginal effects on blood coagulation

and platelet activation (45, 46).

Of the potential therapeutic agents administered to baboons, eptifibatide was the most

effective platelet antagonist, inhibiting platelet aggregation by all agonists tested.

This effect was related to the dose of the drug administered (0-48% iuhibition at 2.0

fLglkg/min. and 84-99% iuhibition at 7.5 fLg/kg/min.). The effect was rapidly induced,

requiring administration for only 30 minutes, and readily reversible, returning to near

baseline values within 2 hours after cessation. As expected, heparin inhibited the

aggregation of baboon platelets by thrombin alone, as heparin catalyzes thrombin

degradation by associating with anti-thrombin. Methylprednisolon.e inhibited ADP

induced platelet aggregation by as much as 55%, an effect that, to our knowledge, has

not been reported previously, but that could relate to membrane stabilization and the

inhibition of phospholipases (40). In contrast to results in other animal models (36,

37), PGI2 did not iuhibit platelet aggregation substantially. This may indicate that

this preparation ofPGI2 is less effective in baboons than in humans. Alternatively, as

PGI2 also acts on the endothelium, the antiplatelet properties of this compound may

require the presence of a functioning endothelium and thus may not be apparent in

this model of platelet aggregation.

When PRP was prepared from baboons treated with eptifibatide, PBPC-induced in

vitro platelet aggregation was inhibited by > 70%. This suggests that eptifibatide, or

other GPIIb/IIIa antagonists, would be beneficial adjuncts to the conditioning regimen

currently used. For this reason, trials investigating the role of eptifibatide in baboons

receiving porcine PBPC have begun. Furthermore, PBPC-induced platelet

aggregation was also iuhibited (by 55%) in PRP prepared from a baboon 6 days after

administration of CPP. The ability of CPP to abrogate PBPC-induced platelet

174


aggregation provides further evidence that CPP may protect baboons receiving PBPC

against TM.

The experimental model of platelet aggregation used in the present study provides

considerable information on the ability of various agents to alter the threshold for

platelet activation and aggregation in baboons. Most molecular interactions would

continue to occur in vivo after the administration of each agent or procedure prior to

withdrawal of blood samples. Additionally, since the PRP subsequently prepared

contains most of the components of blood, many molecular interactions would also

occur during in vitro assays. By inducing an element of low shear stress, the platelet

aggregometer mimics flow conditions in the venous (but not the arterial) vasculature

to some extent. However, this model is limited in that it does not account for the

contributions imparted by the endothelium (whether intact and functional or in a

pathological state) including cell-cell interactions, the release of soluble mediators,

and vasoconstrictive/vasodilatory responses. Thus, in future endeavors, it would be

useful to utilize assays that better assess the role of the endothelium, such as intravital

video microscopy.

Althougb this study demonstrates that porcine PBPC initiate the fonnation of baboon

platelet aggregates in vitro, resulting in thrombocytopenia when infused in vivo, it

does not elucidate which component of the leukapheresis product is primarily

responsible. Flow-activated cell sorting studies demonstrate that the PBPC

leukapheresis product (after one freezing/thawing cycle) contains approximately 30%

T cells, 45% B cells and monocytes, 20% debris (e.g. dead cells and cellular

fragments), and <1 % granulocytes. Any of these cellular subsets, or even soluble

factors that they have released, could contribute to this phenomenon. To determine

whether or not specific cellular subsets of porcine PBPC are responsible for TM, in

vitro studies have begun to assess which, if any, specific fractions induce platelet

aggregation and/or endothelial cell activation. It may be that aggregation is induced

by a fraction that is unnecessary for the engraftment of porcine hematopoietic

progenitor cells, in which case the removal of this subset from PBPC may eliminate

175


the morbidity currently associated with PBPC infusion and facilitate mixed

chimerism.

Substantial PBPC-induced platelet aggregation has only been observed between

discordant species in our laboratory. The addition of baboon PBPC to baboon PRP,

for example, does not result in substantial platelet aggregation (see Chapter 6). Thus,

it will be important to determine if immunological factors or molecular barriers

contribute. It is unlikely that aggregation is mediated by baboon complement since

the depletion of complement by CVF has no effect on the course of TM. Likewise, it

is improbable that preformed xenoreactive antibodies play a major role since the

depletion of anti-Gal (by >95%) by EIA does not affect PBPC-induced platelet

aggregation (Appel, J.Z., et a!. - unpublished observations). It may be that specific

porcine PBPCHbaboon platelet ligands interact more extensively than species-specific

ligands and therefore result in more substantial platelet aggregation. Specific

inhibition of these interactions (e.g. by CPP or GPIIb/IIIa antagonists) may abrogate

this syudrome.

Nevertheless, it is likely that the TM observed in baboons receiving PBPC results

from a multifactorial process. It is possible that the combination of some components

of the conditioning regimen exacerbate TM in baboons. Although platelet

aggregation is not increased after treatment with the full \VBIHbased conditioning

regimen (without PBPC) (Alwayn, I.P.J. - unpublished observations), these baboons

may exhibit a dysfunction in platelet-endothelial interactions not detected in this in vitro model. Furthermore, it is likely that the mechanisms involved in TM extend

beyond simple platelet-platelet and platelet-leukocyte interactions. It will be

important to assess the tendency of porcine PBPC and of PBPC-induced platelet

aggregates to activate primate endothelial cells and studies to invesTigate this have

already been initiated.

176


REFERENCES

1. Cooper DKC, Ye Y, Rolf LL Jr., Zuhdi N. The pig as potential organ donor for man. In: Cooper DKC, Kemp E, Reemtsma K, White DJG, eds. Xenotransplantation. lSI ed. Heidelberg, Gennany: Springer, 1991: 481.

2. Sachs DH. The pig as a potential xenograft donor. Vet Immunollmmunolopathol. 1994; 43: 185. 3. Appel JZ Ill, Buhler L. Cooper DKC. The pig as a source of cardiac xenografts. J Cardiac Surg.

(In press). 4. Buhler L, Friedman T. lacomini J, Cooper DKC. Xenotransplantation - state of the art - update

1999. Frontiers in Bioscience. 1999: 4: d416. http://www.bioscience.org/1999/v4/d/buhlerifulltext.htm

5. Sykes M, Sachs DH. Xenogeneic tolerance through hematopoietic cell and thymic transplantation. In: Cooper DKC, et ai, eds. Xenotransplantation. 2nd ed. Heidelberg, Gennany: Springer; 1997: 496.

6. Kawai T, Poncelet A, Sachs DH, et al. Long-term outcome and alloantibody production in a nonmyeloablative regimen for induction of renal allograft tolerance. Transplantation. 1999; 68: 1767.

7. Buhler L, Basker M, Alwayn IPJ, et a1. Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Transplantation. (In press).

8. Colby C, Chang Q, Fuchimoto Y, et al. Cytokine-mobilized peripheral blood progenitor cells for allogeneic reconstitution of miniature swine. Transplantation. 1999; 69: 135.

9. Nash K, Chang Q, Watts A, et al. Peripheral blood progenitor cell mobilizatior, and Jeukapheresis in pigs. Lab Anim Sci. 1999; 49: 645.

10. Kozlowski T, Monroy R, Giovino M, et a!. Effect of pig-specific cytokines on mobilization of hematopoietic progenitor cells in pigs and on pig bone marrow engraftment in baboons. Xenotransplantalion. 1999; 6: 17.

II. Pettit AR, Clark RE. Thrombotic microangiopathy following bone marrow transplantation. Bone Marrow Transplant. 1994; 14: 495.

12. Bimalangshu 0, Sykes M, Spitzer TR. Outcomes of recipients of both bone marrow and solid organ transplants. Medicine. 1998; 77: 355.

13. Perez-Sanchez I, Anguita J, Pintado T. Use of cyclophosphamide in the treatment of thrombotic thrombocytopenia purpura complicating systemic lupus erythematous: report of two cases. Ann Hematoi. 1999; 78: 285.

14. Ruggenenti P, Remuzzi G. Thrombotic thrombocytopenia and related disorders. Hem. - Onc. Clin. N. Amer. 1990: 4: 219.

15. Strutz F, Wieneke U, Braess J, et al. Treatment of relapsing thrombotic thrombocytopenia purpura with cyclophosphamide pulse therapy. li/ephrology, Dialysis, Transplantation. 1998; 13: 1320.

16. Nomura M, Okada J, Tateno S, et al. Renal thrombotic microangiopathy in a patient with rheumatoid arthritis and antiphosphoJipid syndrome: successful treatment with cyclophosphamide pulse therapy and anticoagulant. Int. Med. 1994; 33: 484.

17. Cooper DKC, Ye Y, and Niekrasz M. Heart transplantation in primates. In: Cramer DV, Podesta L, and Makowka L, eds. Handbook of Animal Models in Transplantation Research. Boca Raton: CRC Press. 1994: 173.

18. Hallahan DE, Virudachalam S. Ionizing radiation mediates expression of cell adhesion molecules in distinct patterns within the lung. Cancer Res. 1997; 57: 2096.

19. Hallahan DE, Kuchibhotla J, Wyble C. Cell adhesion molecules mediate radiation-induced leukocyte adhesion to the vascular endothelium. Cancer Res. 1996; 56: 5150.

20. Phillips TL. An ultrastructural study of the development of radiation injury in the lung. Radiology. \966; 87: 49.

21. Sporn LA, Rubin P, Marder YJ, Wagner DO. Irradiation induces release of von willebrand protein from endothelial ce!ls in culture. Blood. 1984: 64: 567.

22. In: Schrefer J, et al., eds. Mosby's GenRx, 10th ed. St. Louis, Missouri: Mosby. 2000. 23. XU Y, Lorf T, Sablinski T, et al. Removal of anti-porcine natural antibodies from human and

nonhuman primate plasma in vitro and in vivo by a Gal 1-3GaH31-4I3G!c-X immunoaffinity column. Transplantation. 1998: 65: 172.

177


24. Kozlowski T, Shimizu A, Lambrigts D, et a1. Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody absorption. Transplantation. 1999; 67: 18.

25. Blaheta RA., Nelson K, Opperman E, et al. Mycophenolate mofetil decreases endothelial prostaglandin E2 in response to allogenic T cells or cytokines. Transplantation. 2000; 69: 1977.

26. Kobayashi T, Taniguchi S, Neethling FA, et a!. Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy. Transplantation. 1997; 64: 1255.

27. Leventhal JR. Dalmasso AP, Cromwell JW, et al. Prolongation of cardiac xenograft survival by depletion of complement. Transplantation. 1993; 55: 857.

28. Candinas D, Lesnikoski B, Hancock W, et al. Inhibition of platelet integrin GPIIb!IIa prolongs survival of discordant cardiac xenografts. Transplantation. 1996; 62: I.

29. Kawai, T, Andrews D, Colvin RB, Sachs DH, Cosimi AB. Thromboembolic complications after treatment with monoclonal antibody and CD40 ligand. ]I/ature Med. 2000,6: 114.

30. Kirk, A.D., Harlan, D.M. Reply to Kawai, T., et al. Nature Med. 2000,6: 114. 31. Biogen, Inc. Biogen says it has halted several tirals of anti-CD40 ligand monoclonal antibody (Press

release). http://W\v\v.biogen.comlindex2.html. Oct. 21,1999. 32. Henn V, Slupsky JR, Grafe M, et a!. CD40 ligand on activated platelets triggers an inflammatory

reaction of endothelial cells. jVature. 1998; 391: 591. 33. Karolak L, Chandra A, Khan W, et al. High-dose chemotherapy-induced platelet defect: inhibition

of platelet signal transduction pathways. Mol. Pharm. 1993; 43: 37. 34. Panella n, Peters W, White JG, Hannun VA, Greenberg CS. Platelets acquire a secretion defect

after high-dose chemotherapy. Cancer. J 990; 65: 1711. 35. Dormandy JA. Rationale for antiplatelet therapy in patients with atherothrombotic disease. Vase

Med. 1998; 3: 253. 36. Whittle BJ, Moncada S, Vane JR. Comparison of the effects ofprostacyclin (PGI2), prostaglandin

Eland D2 on platelet aggregation in different species. Prostaglandins. 1978; 16: 373. 37. Nishimura H, Naritomi H, Iwamoto Y. et a!. In vivo evaluation of antiplatelet agents in gerbil

model of carotid artery thrombosis. Stroke. 1996; 27: 1099. 38. Boneu B. New antithrombotic agents for the prevention and treatment of deep vein thrombosis.

Haemostasis. 1996; 26: 368. 39. Sakuragawa N. Regulation of thrombosis and hemostasis by antithrombin. Semin Thromb Hemosl.

1997; 23: 557. 40. Samuelsson B, Dahler SE, Lindgren JA, Rouzer CA, Serhan CN. Leukotrienes and lipoxins:

structures, biosynthesis, and biological effects. Science. 1987; 237: 1171. 41. Kleiman NS, Lincoff AM, Flaker GC, et al. Early percutaneous coronary intervention, platelet

inhibition with eptifibatide, and clinical outcomes in patients with acute coronary syndromes. PURSUIT Investigators. Circulation. 2000; 101: 751.

42. Stringer KA. The evolving role of platelet glycoprotein IlbiHIa inhibitors in the management of acute coronary syndromes. Ann. Pharmacother. 1999; 33: 712.

43. Spitzer TR, Delmonico F, Tolkoff-Rubin N, et al. Combined histocompatibility leukocyte antigenmatched donor bone marrow and renal transplantation for multiple myeloma with end stage renal disease: the induction of allograft tolerance through mixed lymphohematopoietic chimerism. Transplantation. 1999; 68: 480.

44. Levine PH. The effect of throiIlbocytopenia on platelet aggregation. Am. J. Clin. Pathol. 1976; 65,79.

45. Loftus JC, Smith JW, Ginsberg, MH. Integrin-mediated cell adhesion: the extracellular face. J. Bio!. Chem. 1994; 269: 25235.

46. Marguerie GA, Edington TS, Plow EF. Interaction of fibrinogen with its platelet receptor as part ofa multistep reaction in ADP-induced platelet aggregation. J. Bioi. Chem. 1980,255: 154.

178

8 Pharmacologic inhIbition of platelet aggregation in baboons

Adaptedfrom: Alwayn IPj*, Appel JZ III', Goepfert C, Buhler L, Cooper DKC, and Robson SC. Inhibition of platelet aggregation in baboons: therapeutic implications for xenotransplantation. Xenotransp/antatian 2000;7(4)247-257 • Authors contributed equally.

PUBMED-ID

HYPERLINK "/pubmed/11081759"Inhibition of platelet aggregation in baboons: therapeutic implications for xenotransplantation. Alwayn IP, Appel JZ, Goepfert C, Buhler L, Cooper DK, Robson SC. Xenotransplantation. 2000 Nov;7(4):247-57. PMID: 11081759 [PubMed - indexed for MEDLINE]


ABSTRACT

Introduction. Activation of endothelial cells and platelet sequestration playa major

role in rejection of xenografts. The histopathology of both hyperacute and acute

vascular or delayed rejection of vascularized discordant xenografts is characterized by

interstitial hemorrhage and intravascular thrombosis. Agents that prevent platelet

activation and consequent microthrombus formation have proven beneficial in

xenograft rejection but do not fully preclude vascular thrombosis. Recently, several

new anti platelet therapies have undergone extensive clinical testing for atherosclerotic

thrombotic vascular disorders; other putative therapies are undergoing preclinical

evaluation. We have investigated the effect of several of these novel agents on platelet

aggregation in baboons in order to screen for future potential in xenograft rejection

models.

Methods. Drugs tested in these experiments were aurintricarboxylic acid (ATA, von

Willebrand Factor-GPIb inhibitor). fucoidin (selectin-inhibitor), l-benzylimidazole

(1-BI, thromboxane synthase antagonist), prostacyclin (POI,. endothelial stabilizer),

heparin (thrombin antagonist), nitroprusside sodium or nicotinamide (NPN or NA,

both NO-donors). and eptifibatide (EFT, GPIIb/lIIa receptor antagonist). These were

infused intravenously to 9 baboons. Coagulation parameters and platelet counts were

monitored and baboons were observed for adverse side effects. Ihe efficacy of these

agents in inhibiting platelet aggregation was assayed in a platelet aggregometer.

Results. Treatment with ATA and fucoidin resulted in complete inhibition of platelet

aggregation but also in major perturbation of coagulation parameters. 1-BI and PGI2

had no effect when administered alone, but in combination resulted in moderate

inhibition of aggregation without disturbance in PI or PTT. NPN and NA had no

substantive effects on platelet aggregation. Heparin resulted in specific inhibition of

thrombin-induced platelet aggregation and, as anticipated, was associated with

moderate prolongation of PIT. Importantly, EFT caused complete inhibition of

platelet aggregation without changes in coagulation. Platelet counts, fibrinogen levels,

and fibrinogen degradation products remained within the normal ranges in all

experiments.

180

Pharmacotherapy and platelet aggregation

Conclusions. Although excellent inhibition of platelet activation was obtained with

AT A and fucoidin, clinical use may be precluded by concomitant disturbances of

coagulation. Combinations of heparin and EFT may prove beneficial in preventing the

thrombotic disorders associated with xenograft rejection while maintaining adequate

hemostatic responses. These agents are to be evaluated in our pig-to-primate

xenotransplantation models.

INTRODUCTION

Transplantation of vascularized porcine organs in untreated primates results in

hyperacute rejection within minutes to hours after reperfusion (1,2,3). "When

hyperacute rejection is averted, either by depletion of xenoreactive antibodies or

complement or by using donors transgenic for human complement-regulatory

proteins, rejection usually occurs within days to weeks and is termed acute vascular or

delayed xenograft rejection (4,5,6,7). All fonus of rejection <ire associated with

vascular il~ury resulting at least in part from platelet activation (8,9,10,11,12).

Rejected xenografts invariably demonstrate intravascular thrombosis and interstitial

hemorrhage (2,3,4,13,14). In addition, following miniature swine-to-baboon kidney

transplantation, disseminated intravascular coagulation has developed within 6-10

days. This process had been manifest by steadily decreasing fibrinogen and platelet

counts, with increased prothrombin time (PT) and fibrinogen degradation products

(15,16,17). This state has developed before histopathological evidence of rejection

was advanced (17).

In our attempts to induce immunological tolerance in the miniature swine-to-baboon

model by achieving mixed hematopoietic chimerism, a thrombotic microangiopathic

state has been observed after the systemic infusion of mobilized porcine peripheral

blood progenitor cells. Thrombocytopenia, high levels of lactate dehydrogenase, and

erythrocyte schistocytosis have been observed (17,18).

The potential benefit of antithrombotic agents in the treatment of transplantation

rejection is well recognized, and has been the subject of investigation for numerous

181


years (9,19,20,21,22), Inhibition of platelet activation, recruitment, and sequestration

might inhibit development of the thrombotic microangiopathy and/or of the

disseminated intravascular coagulation. There have been major advances in

understanding mechanisms of platelet activation that have resulted in the development

of new therapeutic strategies to block the formation of platelet microthrombi (9,23).

We have therefore investigated the effect of various agents on platelet aggregation.

Eight drugs were selected and then administered to baboons by intravenous (iv)

infusion; the effect of each agent on platelet aggregation was tested in vitro.

MATERIALS AND METHODS

Animals

Baboons (Papio Anubis, n=9, Biomedical Resources Foundation, Houston, TX),

weighing approximately 9-15 kg, were used for testing the experimental agents. All

animals had been previously splenectomized through a midline laparotomy. Five

baboons had previously been treated with a regimen aimed at inducing immunological

tolerance, either with or without infusion of mobilized porcine peripheral blood

progenitor cells> 4 months prior to the current experiments (15). Three baboons had

previously (>3 months) received pharmacologic immunosuppressive therapy. Each

baboon was administered at least two of the agents tested, but coagulation parameters

and platelet aggregation were allowed to return to baseline before a second drug was

tested.

All experiments were performed in accordance with the Guide for the Care and Use of

Laboratory Animals prepared by the National Academy of Sciences and published by

the National Institutes of Health. Protocols were approved by the Massachusetts

General Hospital Subcommittee on Research Animal Care.

Surgical Procedures

Baboons were given atropine (0.05 mg/kg im) and ketamine (10 mg/kg im) as

premedication, and were intubated and ventilated through a closed circuit with oxygen

(1-2 Limin), nitrous oxide (2-4 Llmin), and isoflurane (0.5-3.0 %). The baboon's

182


carotid artery and internal jugular vein were cannulated, the cannulae were tunneled

subcutaneously towards the scapula, and brought out through a jacket and tethering

system. This allowed monitoring of arterial blood pressure, access to blood

withdrawal, and continuous or intermittent iv administration of fluids and/or drugs.

Cefazolin (500 mg iv daily) was given as prophylaxis against infection.

Experimental Agents

All agents, or combination of agents, were tested in at least two baboons, and were

administered through continuous iv infusion unless otherwise described. Bolus

injections are specified when used.

Aurintricarboxylic acid (ATA, Sigma-Alldrich, St. Louis, MO), administered at 0.5-

8.0 mg/kgihour, is kuown to inhibit binding of von Willebrand factor (vWF) to

platelet glycoprotein Ib (GP Ib) in vitro and has been shov.TI to significantly inhibit

platelet adhesion and platelet aggregation in several small animal models (24,25,26).

The observed anti thrombotic activity possibly arises from inhibitory effects on

thrombin activity and the vWF-GPlb pathway.

Fucoidin (Fluka Chemie AG, Buchs, Switzerland), administered at 0.5 - 1.0

mg/kglhour, is a non-toxic sulfated fucose oligosaccharide derived from seaweed that

blocks platelet (P)-selectin and may prevent platelet adhesion and associated

aggregation responses (27,28,29,30). Selectins are adhesion molecules found on

leukocytes (L-selectin), endothelium (P- and E-selectin), and platelets (P-selectin) that

bind to oligosaccharide ligands containing fucose and sialic acid to mediate leukocyte

rolling on, and platelet adhesion to, the endothelium. Inhibition of these selectins may

result in reduced platelet adhesion andlor aggregation (29,30).

Thromboxane A2, an enzymatic product of arachidonic acid, has been implicated in

platelet aggregation and the initiation of vasoconstriction in various organ systems

(31,32). Selective inhibition of thromboxane synthase, which converts prostaglandin

H2 to fhromboxane A2, using l-benzylimidazole (I-BI, Cayman Chemical Company,

AIm Arbor, MI) has been shown to reduce thromboxane A2 concentration in vivo in

183


rat brains (33). l-BI was administered at 10 - 40 Ilg/kg/hour as a single agent or in

conjunction with POh (see below).

Prostacyclin (PGI" Glaxo Wellcome Inc, Research Triangle Park, NC) is an

endothelial-derived vasodilator with reported anti-thrombotic effects (34). It has been

documented to inhibit platelet aggregation in vitrQ in small animal models (35,36).

PGh was administered at 20 - 40 ng/kg/min as a single agent, and in conjunction with

l-BI at 30 ng/kg/min.

Heparin has well-known actions in potentiating antithrombin III and thereby

influencing thrombin-mediated platelet aggregation (37,38). Thrombin catalyzes the

formation of fibrin from fibrinogen, reSUlting in thrombus formation, and strongly

induces platelet activation and recruitment (39). Furthermore, thrombin promotes

chemotaxis by monocytes, and stimulates P-selectin activity in endothelial cells (40).

Heparin sodium (Heparin, Elkins-Sinn, Inc., Cherry Hill, NJ) was administered at 10

- 50 Units/kglhour.

Nitroprusside sodium (NPN, Ohrneda PPD Inc., Liberty Comer, NJ), administered at

1.0 - 10 Ilg/kg/min, is a well-<lescribed nitric oxide (NO)-donor that may also have

an inhibitory effect on platelet aggregation. NO, formally referred to as the

endothelium-derived relaxation factor, is a potent vasodilator that maintains

endothelial integrity (41,42). It has also been described to have anti-platelet effects

through increasing the intracellular cyclic guanidine monophosphate in platelets (43).

Nicotinamide (NA, Sigma Chemical Co., St, Louis, MO), administered at 0.25 - 0.5

mg/kglhour, is a B-complex vitamin that is also associated with increased NO

production and vasodilatation (44). NA may therefore be effective in inhibiting

platelet aggregation for the same reasons as NPN.

Eptifibatide (EFT, Cor Therapeutics, Inc., South San Francisco, CAl was

administered as a bolus infusion of 180 Jlglkg iv followed by continuous iv infusion at

2.0 - 7.5 Ilg/kg/min. EFT is a synthetic GP Ilb/Illa receptor antagonist currently used

in the treatment of acute myocardial infarction and for post-angioplasty restenosis

184


(45,46). By preventing GP lIb/IlIa receptor binding to fibrinogen, crosslinking of

platelets is precluded and aggregation is specifically inhibited.

Coagulation Parameters

Serial blood samples were taken for measurement of platelet count, prothrombin time

(PT), partial thromboplastin time (PTT), fibrinogen, and fibrinogen degradation

products (FDP) (Hematology Laboratory, Massachusetts General Hospital).

Platelet Aggregation Assay (Figure 1)

Ten milliliters citrated blood were drawn from the baboon and centrifuged at 280g for

15 minutes at 22°C. The supernatant, consisting of platelet-rich plasma, was

transferred in 400 J.lI aliquots to siliconized glass cuvettes, and formed the

experimental samples. The platelets were counted in a Coulter counter (Beckman

Coulter, Inc., Fullerton, CA) and, if necessary, the experimental samples were diluted

to a final concentration of lxl08 platelets/ml with a calcium-, magnesium-, and

phenol red-free Hanks buffer solution (Mediatech, Inc., Herndon, V A). The reference

sample, consisting of platelet-poor plasma, was used to set the baseline level of

aggregation to 0%. This was obtained by further centrifuging the remaining pellet at

2,400g for 2 minutes at 22°C.

Different platelet agonists/stimulators (agents that are known to initiate platelet

aggregation) were added to the experimental samples in the platelet aggregometer

(two-sample, four-channel, model 560 Ca Lumi-aggregorneter, Chronolog Corp.,

Havertown, PA) at set concentrations, and aggregation was induced with a magnet

causing shear stress. The agonists used were thrombin (1 - 5 U/ml, causing platelet

activation and aggregation by binding to platelet GP Ib and cleaving platelet GP V),

185


Figure I.

Citra ted blood

n 280g for 15 minutes

Platelet-rich plasma = n 2,400g for 2 minutes

Experimental samples

Platelet-poor plasma (reference sample)

= Aggregometer (baseline)

Addition of platelet ~ ~ .•.... agoDlsts ~ ~

Aggregation profiles

Platelet aggregation assay. Citrated blood collected from baboons was centrifuged for 15 minutes at

280g at room temperature. The supernatant, platelet-rich plasma, was then used as experimental

samples. Changes in optical density after adding platelet agonists (thrombin, collagen, ADP, ristocetin,

and arachidonic acid) were measured in the aggregometer against the optical density reference sample,

platelet-poor plasma (see methods).

adenosine diphosphate (ADP) (5 - 20 flM, inducing platelet shape change and

aggregation by binding to multiple high-affinity ADP-specific receptors on platelets),

collagen (5 - 10 )..tg/ml, inducing platelet aggregation by interacting with multiple

platelet receptors such as GP Ib and fibronectin), ristocetin (1.25 ).lg/ml, initiating

platelet aggregation by interacting with the GP Jb-vWF binding site). Arachidonic

acid (1.2 rnJ\1) was substituted for ristocetin when an agent was tested that was not

expected to interact with the GP Ib receptor. Platelet aggregation was measured as a

percentage change in light optical density from baseline (with baseline being the

measurement obtained in the experimental sample in the absence of the platelet

aggregation agonist when compared with the reference sample (see Fig. 1)). Typical

aggregation curves obtained from a naIve, untreated baboon are depicted in Fig. 2.

186


A B

c D

Figure 2.

Control baboon platelet aggregation curves. Agonists were added to platelet-rich plasma in the

aggregometer. Results are from a representative experiment. Initial increase in percentage aggregation

is due to shape changes of activated platelets.

A. Addition of thrombin resulted in 68% aggregation.

S. Addition of collagen led to 48% aggregation.

C. Addition of ADP led to 53% aggregation.

D. Addition ofristocetin resulted in 68% aggregation.

RESULTS

The clinical results and in vitro assays are summarized in Table 1. Coagulation

parameters (PT, PTT, fibrinogen, and circulating FDP levels), platelet count and

aggregation profiles were all within normal ranges at the onset of each experiment,

and none of the agents tested induced any changes in platelet count, fibrino gen, or

FDP levels.

187


Aurintricarboxylic acid (AT A)

ATA was administered in increasing doses from 0.5- 8.0 mg/kglhour iv. At a

cumulative dose of 15 mg/kg, vomiting was observed as well as an increased heart

rate to >200 bpm with a simultaneous drop in blood pressure to 65/35 mmHg.

Cessation of the drug resulted in rapid recovery. When re-administered after an

interval of several days at 0.5 mg/kglhour, no effect on platelet aggregation or

changes in coagulation were observed (data not sho\Vll). Increasing the dose to 1.0

mg/kg/hour resulted in decreased platelet aggregation induced by ristocetin (Fig. 3 -

left). This was accompanied by a prolongation in PT from 14.0 to >50 seconds (Fig. 3

- right). Discontinuing the drug resulted in recovery of the coagulation parameters

within 3 days, but one baboon died from intrawabdominal bleeding 2 days after

cessation of the drug. Infusion of ATA at 2.0 mg/kglhour completely abrogated

aggregation induced by ristocetin (Table I). The other agonists (i.e. thrombin,

collagen, and ADP) were not influenced by ATA therapy.

Fucoidin

Fucoidin at 0.5 ~glkg/hour did not alter the platelet aggregation or coagulation

profile. At 1 !-lg/kg/hour for 4 hours, nearwcomplete inhibition of aggregation induced

by all agonists was achieved (Table 1). This also resulted in an increase in PT from 18

to 65 seconds. No other side effects were documented and recovery of coagulation

parameters occurred rapidly following discontinuation of the drug.

I-benzylimidazole (I-BI)

I-BJ at 20 or 40)lg/kglhour did not substantively change platelet aggregation induced

by any of the platelet agonists (Table I). Coagulation parameters remained

unchanged, and there were no untoward side effects.

Prostacyclin (PGI,)

Inconsistent changes III platelet aggregation were observed with this agent. The

maximum inhibition of aggregation obtained with PGh was 40 and 70% for ADP and

arachidonic acid, respectively (Table 1). No changes in coagulation parameters and no

additional toxic effects were observed at doses between 10- 50 ng/kg/min.

188

TABLE 1

CHANGE IN PLATELET AGGREGATION AND COAGULATION PARAMETERS IN BABOONS

Number Inhibition of Platelet Disturbance of Comments

of Baboons Aggregation Coagulation

Thrombin ADP Collagen Ristocetin Arach Parameters

acid -,--- -

~------- Clinical bleeding, major ATA 2 + + ++ +++ N/A Yes

increase in PT. --~--

Fucoidin 2 +++ +++ +++ ++ N/A Yes Major increase in PT. ---------

J-BI 4 - - - - N/A No -- ------------

PGI2 2 - - - N/A ++ No -

l-BI + PGI, 2 + - - - N/A No Moderate inhibition of platelet

aggregation. -----

Heparin 2 +++ - - - N/A Yes Anticipated increase in PTT. -- -------

NPN 2 + - - N/A + No -f-= .... ---------

NA 2 - - - N/A + No -

EFT 2 +++ +++ +++ N/A +++ No Dose-dependent inhibition of

platelet aggregation by all

I agonists.

Average percentage inhibition of platelet aggregation: - = 0-20; + = 20-50; ++ = 50-80; +++ = >80. N/A: nol applicable


Figure 3.

Aurintricarboxylic acid (AT A)

ATAdsoontinued

01 0 o 1 2 3 4 5 6 7 8

~- "'" • ATA1.0rrglkg'h ti ATA20 rrgtkgIh

--~--PTT

-PT

Left. Administration of AT A at 1.0 mg/kg/h and 2.0 mg/kglh by continuous iv infusion. A dose

dependent inhibition of aggregation induced by ristocetin and, to a Jess extent, by collagen was

achieved, with little response to ADP and thrombin.

Right. At an AT A infusion of 2.0 mglkglh, an increase in PT and PIT to >50 and >150 seconds,

respectively, was observed. Cessation of AT A led to recovery of coagulation parameters.

I-benzylimidazole (I-BI) and prostacyclin (PGI,)

The combination of I-BI (at 10 j.lglkglhour) with PGI, (at 30 ng/kg/min) in one

baboon resulted in progressive blockade of platelet aggregation induced by thrombin.

Increasing I-BJ to 20 j.lg/kglhour led to complete inhibition of aggregation induced by

thrombin. Aggregation induced by other agonists was not significantly changed

(Table I). A further increase of I-BJ to 40 j.lglkglhour in a second baboon, however,

resulted in only minimal inhibition of platelet aggregation. In combination, the two

agents did not result in any substantial changes in coagulation parameters.

Heparin

Heparin at 10 U/kg/hour led to a predicted increase in PIT from 29 seconds to 100

seconds in one baboon, and was associated with complete inhibition of platelet

aggregation induced by thrombin (Table I). Aggregation profiles induced by collagen,

ristocetin, and ADP remained unchanged. In a second baboon, however, heparin at 10

190


U/kg/hour did not alter the coagulation parameters nor inhibited platelet aggregation.

At an incremented dose of 50 U/kglhour, an increase in PTT to 100 seconds was

observed. This was accompanied by inhibition of aggregation induced by thrombin.

No clinical signs of bleeding or other side effects were observed at this time and

heparin dosage.

Nitroprusside sodium (NPN)

NPN (at 1.0 - 15 ~g/kg/min) resulted in a modest decrease in platelet aggregation

induced by thrombin and arachidonic acid in one baboon, but did not affect

aggregation in a second baboon, suggesting individual sensitivity (Table 1). At doses

higher than 10 J..lglkglmin, vomiting was observed without changes in blood pressure

or coagulation parameters.

Nicotinamide (NA)

NA (at 0.25 - 0.5 mg/kglhr) did not cause significant inhibition of platelet

aggregation although aggregation induced by arachidonic acid was decreased to some

extent (Table 1). No other effects of the drug were noted.

Eptifibatide (EFT)

EFT was administered as an iv bolus of 180 J..lglkg followed by continuous infusions

at dosages ranging from 2.0 -7.5 ~g/kglmin. Platelet aggregation induced by all

agonists used (i.e., thrombin, ADP, collagen, and arachidonic acid) was effectively

inhibited with increasing dosages of EFT (Table 1 and Fig. 4). EFT at 2.0 f!g/kg/min

had no effect on aggregation. Increasing the dose to 5.0 and 7.5 f!g/kg/min led to a

dose-dependent complete inhibition of aggregation induced by ADP (Fig. 5) and by

other agonists. Cessation of the drug led to full recovery of platelet aggregation within

2 ~ 4 hours. During treatment with EFT, no side effects were noted, and no changes in

coagulation parameters were observed.

191


OThrombin

!l!IADP

OColiagen

"",IO,'m'" mo,,,,,",,,

Agent {dosol

Figure 4.

Eptifibatide (EFT). EFT effectively inhibited platelet aggregation in a dose-dependent manner. At 2

).1glkglmin, no response was noted (data not shown). At 5 !Jog/kg/min, aggregation responses induced by

thrombin, ADP, collagen, and arachidonic acid decreased markedly. EFT when administered at 7.5

).1g/kg/min resulted in a further, near-complete decrease in aggregation induced by all agonists. Two

hours following discontinuation of EFT, aggregation returned to baseline levels.

DISCUSSION

There are two major reasons related to xenotransplantation why we have investigated

drugs that inhibit platelet aggregation.

Firstly, in our experience, the histopathological features of hyperacute and acute

vascular rejection of vascularized xenografts in the pig-to-nonhuman primate model

invariably demonstrate intravascular thrombosis and interstitial hemorrhage.

Miniature swine kidney transplants into baboon recipients frequently show features of

disseminated intravascular coagulation, even before rejection is histopathologically

advanced (15,16,17). We believe that activation of recipient platelets and/or donor

vascular endothelium play major roles. Prevention of these thrombotic events may

extend xenograft survival by preserving adequate perfusion and maintaining

192


oxygenation of the graft even though the underlying inflammatory mechanisms may

not be fully prevented.

Secondly, the transplantation of mobilized porcine peripheral blood progenitor cells to

baboons in an attempt to induce immunological tolerance through mixed

hematopoietic chimerism has been associated with a thrombotic microangiopathy,

characterized by thrombocytopenia (platelet count < 20,000/f!1), elevated lactate

dehydrogenase, and erythrocyte schistocytosis (18). In our laboratory, we have

observed the formation of baboon platelet aggregates in the presence of mobilized

porcine peripheral blood progenitor cells in vitro (Alwayn IPJ, et aI., unpublished

data). Drugs that modulate formation of these aggregates may allow the infusion of

more porcine progenitor celis, thus facilitating engraftment of these cells and the

development of mixed hematopoietic chimerism.

It has become clear that the binding of agonists/stimulators to receptors on the platelet

surface plays a central role in inducing platelet activation. Platelet adhesion to

subendothelium leads to activation of the platelets) after which platelet sequestration

and formation of aggregates can occur. VVhen circulating platelets come into contact

with exposed subendothelium (i.e. after endothelial cell retraction), adhesion occurs

through binding of subendothelial platelet agonists, such as vWF, to platelet GP Ib

under shear stress (47,48). This results in activation of platelets with

release/generation of more platelet mediators, such as thrombin, ADP, and

arachidonic acid (a precursor of thromboxane A2). These agonists generally have

their own specific receptors on platelets and cause the platelets to change shape, form

pseudopodia, and activate the GPIIb/IIIa receptor on the platelet surface. Cross

linking of these receptors with fibrinogen or soluble vWF results in platelet

aggregation (49,50). Recent data suggest that platelets may also bind directly to

endothelial cells and subsequently become activated (51). Other platelet receptors

include a2pl integrin, GP IV, and GP VI (all receptors for collagen). Although there

are many diverse substances that can activate platelets, our interest was in testing

agents that interact with platelets at different points of application.

193


Although treatment with AT A or fucoidin was very effective at inhibiting platelet

aggregation, both gave rise to severe disturbances of coagulation parameters, which

may preclude their use in a clinical setting. The effects of AT A and fucoidin on

coagulation parameters were not anticipated and could not directly be explained by

the mechanisms by which these agents interact with platelets. It is, however, possible

that, in addition to inhibiting receptors on platelets, they interact with other

components of the coagulation pathway that have not yet been delineated.

I-BI was brought to our attention by Pierson and his colleagues, who have used

thromboxane synthase inhibitors in a pig-to-human xenoperfusion model in an effort

to prevent pulmonary vasoconstriction (52). This agent has also been sho\Vtl to

improve perfusion in a rat cerebrovascular ischaemia model by preventing the

production ofthromboxane A2, a potent platelet agonist (35). When administered to a

baboon, we observed no significant changes in platelet aggregation even when

dosages in excess of those recommended in other experimental models were used. It

is possible, though unlikely, that therapeutic levels of I-BI necessary to effectively

inhibit platelet aggregation were not achieved. In these experimental settings, no

changes in PT or PTT were observed.

PGh, a metabolite of arachidonic acid, has potent vasodilatory effects on pulmonary

and systemic arterial beds (53,54) and has been reported to be an inhibitor of platelet

aggregation in humans (55). We administered this drug in doses exceeding those

recommended for humans without any significant change in blood pressure, heart rate

or coagulation parameters. In contrast to the human data, no suppression of platelet

aggregation in vitro could be obtained. When PGI2 was combined with a high dose of

1-BI, however, moderate inhibition of platelet aggregation induced by thrombin was

obtained, unaccompanied by changes in PT or PTT.

Heparin has been used in several models in an attempt to prolong survival of

transplanted allo- or xeno-grafts, with varying results (21,56,57). In the latter two

experiments, heparin was effective in preventing platelet aggregation induced by

194


thrombin in vitro. This effect was only observed when the PTT had reached a

therapeutic level of> 1 00 seconds, and was not associated with clinical bleeding.

It has been sho\\11 that NO has protective effects on the endothelium (43,44,45) and

can prevent platelet aggregation by increasing intracellular cyclic guanidine

monophosphate (cGMP) levels in platelets. In our model, however, NPN, an NO

donor, did not lead to any substantive decrease in aggregation. One explanation may

be that insufficient levels of NO were obtained in our dosing regimen, as no

associated vasodilatory effects, such as hypotension, were noted. Furthermore, since

NO has both inflammatory oxidant-mediated effects (58) as well as platelet

aggregation inhibitory potential (45), titrating towards a therapeutic dose will be

challenging. Additionally, exogenous NO may exacerbate inflammation through

synthesis of platelet-activating factor and mobilization of P-selectin (59). NA, also

associated with increased NO production and vasodilatation, may have a similar mode

of action as NO-donors (46). However, we observed no inhibitory effect on platelet

aggregation in the studies presented here. It is possible that endothelial cells may be

more susceptible to NO protection than platelets and that these potentially beneficial

effects of NO may be more apparent in an in vitro model of platelet-endothelium

interaction.

The last and most promising agent tested was EFT. This agent is a synthetic OP

lIb/IlIa receptor antagonist that has been sho\\Tl to have potent inhibitory effects on

platelet aggregation in humans (47,48). We were able to confirm these results in

baboons. EFT was studied instead of the monoclonal antibody abciximab, which also

targets the OP lIb/IlIa receptor, because of the lower incidence of intracerebral

hemorrhage, decreased potential for Fe-interactions, and absent risk for cross

immunization associated with treatment with EFT. No side effects were noted at the

dosages administered. The complete inhibition of platelet aggregation in the absence

of any changes of coagulation parameters may be of benefit in preventing the

thrombotic disorders associated with the transplantation of porcine vascularized

organs or hematopoietic progenitor cells. In the discordant guinea pig-to-rat cardiac

xenograft model Candinas et aL demonstrated prolonged xenograft survival and

195

Section IV ~ Chapter 8

diminution of intragraft platelet micro thrombi using the GPUb/lIla antagonist GPI

562. These results were not confinned in a recent ex vivo discordant perfusion model

of a porcine kidney with human blood using the GPIIb/IIIa antagonist abciximab (60).

In this model, however, as no measures were undertaken to prevent hyperacute

rejection, the effect of inhibition of platelet aggregation post-hyperacute rejection was

not addressed.

We recently administered EFT as an initial iv bolus of 180 f.lglkg, followed by

continuous iv infusion at 7.5 ).lg/kg/min, to one baboon receiving our conditioning

regimen aimed at inducing immunological tolerance. This non-myeloablative regimen

has been described elsewhere (15), and consists of splenectomy, whole body (300

cGy) and thymic (700 cGy) irradiation, multiple extracorporeal irnmunoadsorptions of

anti-aGal antibodies, T cell depletion, phannacological immunosuppressive therapy,

and costimulatory blockade, prior to the infusion of large numbers of mobilized

porcine peripheral blood progenitor cells. The infusion of pig cells is always followed

by an immediate and profound thrombocytopenia that can be ameliorated by steroids,

PGI" and heparin. When EFT was infused, in the absence of heparin, the onset of

thrombocytopenia was delayed by one day, but not prevented. These data suggest

that, although inhibition of platelet-platelet aggregates may provide some benefit,

other factors (e.g. platelet-leukocyte, or platelet-(sub)endothelial interactions)

contribute to these thrombotic complications, and that receptors other than GPIIb/IIla

may also be involved.

In the context of solid vascularized organ xenotransplantation, in one experiment in

our pig-to-baboon model, the combined administration of heparin and EFT appeared

to reverse the early features of disseminated intravascular coagulation (increasing

thrombocytopenia and steadily falling fibrinogen levels) (data not shown).

Other platelet-inhibitors have been described and remam under experimental

evaluation in other models. The vascular NTP-diphosphohydrolase (CD39) is another

candidate for study, as are ADP-receptor antagonists, such as ticlopidine. The lack of

a fully active truncated form of CD39 has delayed testing of this agent, and ADP-

196


receptor antagonists have been implicated in the initiation of thrombotic

thrombocytopenic ptrrpura (61).

These data lead us to conclude that, although AT A and fucoidin are potent inhibitors

of platelet aggregation, the experimental use of these agents in baboons may be

prohibited by an associated high risk of bleeding. The combination of l-BJ and PGJ,

results in moderate inhibition of platelet aggregation, without changes in coagulation

or other side effects, but its effect may be insufficient to be of clinical value. Heparin

leads to good inhibition of thrombin-induced platelet aggregation at doses that cause

only minor prolongation of PTT, allowing for its use in xenotransplantation models.

NPN and NA do not appear to effectively inhibit platelet aggregation in these studies.

EFT clearly was efficient in inhibiting platelet aggregation without any untoward side

effects and will be considered for incorporation into our pig-to-primate transplantation

model for further study.

REFERENCES

1 Perper RJ, Najarian JS. Experimental renal heterotransplantation. I. In widely divergent species. Transplantation 1966; 4: 377

2 Cooper DKC, Human PA, Lexer G, et al. Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the baboon. J Heart LWlg Transplant 1988; 7: 238

3 Kobayashi T, Taniguchi S, Neethling FA, et al. Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy. Transplantation 1997; 64: 1255

4 Parker W, Saadi S, Lin SS, et al. Transplantation of discordant xenografts: a challenge revisited. Immunol Today 1996; 17:373

5 Bach FH, Robson SC, Winkler H, et al. Barriers to xenotransplantation. Nature Med 1995; I: 869 6 Chen RH, Naficy S, Logan IS, et aL Hearts from transgenic pigs constructed with CD59/DAF

genomic clones demonstrate improved survival in primates. Xenotransplantation 1999; 6: 194 7 Marquet RL, van Overdam K, Boudesteijn EA, et al. Immunobiology of delayed xenograft rejection.

Transplant Proc 1997; 29: 955 8 Rosenberg Je, Broersma RJ, Bullemer G, et al. Relationship of platelets, blood coagulation, and

fibrinolysis to hyperacute rejection of renal xenografts. Transplantation 1969;8: 152 9 Robson SC, Schulte am Esch J 2nd

, Bach FH. Factors in xenograft rejection. Ann NY Acad Sci 1999; 875,261

10 Schulte am Esch J 2nd, Cruz MA, Siegel JB, et al. Activation of human platelets by the membraneexpressed Al domain of von Willebrand factor. Blood 1997; 90: 4425

11 Siegel JB, Grey ST, Lesnikoski BA, et al. Xenogeneic endothelial cells activate human prothrombin. Transplantation 1997; 64: 888

12 Koyamada N, Miyatake T, Candinas 0, et al. Apyrase administration prolongs discordant xenograft survival. Transplantation 1996; 62: 1739

J 3 Platt JL, Fischel RJ, Matas Al, et a1. Immunopathology of hyperacute xenograft rejection in a swine-to-primate model. Transplantation 1991; 52: 514

14 Romano E, Neethling FA, Nilsson K, et at. Intravenous synthetic alphaGal saccharides delay hyperacute rejection following pig-to-baboon heart transplantation. Xenotransplantation 1999; 6: 36

197

Sec/ion IV - Chapter 8

15 lerino FL, Kozlowski T, Siegel 18, et al. Disseminated intravascular coagulation in association with the de!ayed rejection of pig-to-baboon renal xenografts. Transplantation 1998; 66: 1439

16 Kozlowski T, Shimuzu A, Lambrigts D, et al. Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption. Transplantation 1999; 67: 18

17 Buhler L, Basker M, Alwayn IPJ, et aJ. Coagul('tion and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Transplantation 2000; 70: 1323

J 8 Buhler L, Goepfert C, Kitamura H, et a!. Porcine hematopoietic cell transplantation in nonhuman primates is complicated by thrombotic microangiopathy. Submitted for publication.

19 Moberg A W, ShoDS AR, Gewurz H, et a1. Prolongation of renal xenografts by the simultaneous sequestration of preformed antibody, inhibition of complement, coagulation and antibody synthesis. Transpl Proc 1971;3:538

20 Ghilchik MW and Morris AS. Platelet trapping by sheep kidney and heart xenografts in the dog. J Surg Research 1974; 1 7:434

21 Kincaid-Smith P. Modification of the vascular lesions of rejection in cadaveric renal allografts by dipridamole and anticoagulants. Lancet 1969;2:920

22 Lesnikoski BA, Candinas D, Otsu I, et al. Thrombin inhibition in discordant xenotransplantation. Xenotransplantation 1997;4: 140

23 Marcus AJ. Pathogenesis of atherosclerosis: special pathogenetic factors-inflammation and immunity: platelets. In Atherosclerosis and Coronary Artery Disease. Vol I. Fuster V, Ross R, and Topol EJ, editors. Lippincott-Raven, Philadelphia 1996: 607

24 lto T, Matsuno H, Kozawa 0, et al. Comparison of the antithrombotic effects and bleeding risk of fractionated aurin tricarboxylic acid and the GPllb/lIla antagonist GRI44053 in a hamster model of stenosis. Thromb Res 1999; 95: 49

25 Kawasaki T, Kaku S, Kohinata T, et al. Inhibition by aurintricarboxylic acid of von Willebrand factor binding to platelet GPlb, platelet retention, and thrombus formation in vivo. Am J Hematol 1994; 47: 6

26 Yamamoto H, Vreys I, Stassen JM, et al. Antagonism ofvWF inhibits both injury induced arterial and venous thrombosis in the hamster. Thromb Haemost 1998; 79: 202

27 Nishino T, Fukuda A, Nagumo T, et at. Inhibition of the generation of thrombin and factor Xa by a fucoidan from the brown seaweed Ecklonia kurome. Thromb Res 1999; 96: 37

28 Millet J, Jouault SC, Mauray S, et al. Antithrombotic and anticoagulant activities of a low molecular weight fucoidan by the subcutanaous route. Thromb Haemost 1999; 81: 391

29 Dardik R, Savion N, Kaufmann Y, et al. Thrombin promotes platelet-mediated melanoma cell adhesion to endothelial cells under flow conditions: role of platelet glycoproteins P-selectin and GPIlb-IlIa. BrJ Cancer 1998; 77: 2069

30 Merhi Y, Provost P, Chauvet P, et al. Selectin blockade reduces neutrophil interaction with platelets at the site of deep arterial injury by angioplasty in pigs. Arterioscler Thromb Vasc Bioi 1999; 19: 372

31 Thomas DW, Mannon RB, Mannon P J, et al. Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2. J Clin Invest 1998; 102: 1994

32 Hirata T, Ushikubi F, Kakizuka A, et al. Two thromboxane A2 receptor isoforms in human platelets. Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation. J Clin Invest 1996; 97: 949

33 Pettigrew LC, Grotta JC, Rhoades I-lM, et al. Effect ofthromboxane synthase inhibition on eicosanoid levels and blood flow in ischemic rat brain. Stroke 1989; 20: 627

34 Dormandy JA. Rationale for antiplatelet therapy in patients with atherothrombotic disease. Vasc Med 1998; 3: 253

35 Wu KK. Endothelial prostaglandin and nitric oxide synthesis in atherogenesis and thrombosis. J Formos Med Assoc 1996; 95: 661

36 Nishimura H, Naritomi H, Iwamoto Y, et aL In vivo evaluation of anti platelet agents in gerbil model of carotid artery thrombosis. Stroke 1996; 27: 1099

37 Boneu B. New antithrombotic agents for the prevention and treatment of deep vein thrombosis. Haemostasis 1996; 26: 368

38 Sakuragawa N. Regulation of thrombosis and hemostasis by antithrombin. Semin Thromb Hemost 1997:23: 557

39 Narayanan S. Multifunctional roles of thrombin, Ann Clin Lab Sci 199; 29: 275

198


40 Coughlin SR. How the protease thrombin talks to cells. Proc Natl Acad Sci 1999; 96: 11023 4 J Vinten~Johansen J, Zhao ZQ, Nakamura M, et a1. Nitric oxide and the vascular endothelium in

myocardial ischaemia-reperfusion injury. Ann N Y Acad Sci 1999; 874: 354 42 Goodwin DC, Landino LM, and Marnett LJ. Effects of nitric oxide and nitric oxide-derived species

on prostaglandin endoperoxide synthase and prostaglandin biosynthesis. FASES J 1999; 13: 1121 43 Moncada S, Palmer R.t\1, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology.

Pharmacol Rev 1991: 43: 109 44 Nishikawa Y, Kanki H, Ogawa S. Differential effects ofN-acety1cysteine on nitroglycerin- and

nicorandil+induced vasodilation in human coronary circulation. J Cardiovasc Pharmacol 1998; 32: 21

45 Kereiakes OJ, Broderick TM, Roth EM, et al. Time course, magnitude, and consistency of platelet inhibition by abciximab, tirofiba, or eptifibatide in patients with unstable angina pectoris undergoing percutaneous coronary intervention. Am J Cardiol 1999; 84: 391

46 The PURSUIT Trial Investigators. Inhibition of platelet glycoprotein IIb/ilia with eptifibatide in patients with acute coronary syndromes. NEJM 1998; 339: 436

47 Body sc. Platelet activation and interactions with the microvasculature. J Cardiovasc Pharmacol 1996; 27: S 13

48 Marcus Al, Safier LB. Thromboregulation: multicellular modulation of platelet reactivity in hemostasis and thrombosis. FASEB J 1993; 7: 516

49 Shattil SJ. Regulation of platelet anchorage and signaling by integrin alpha lIb beta 3. Thromb Haemost 1993; 70: 224

50 Phillips DR, Fitzgerald LA, Charo IF, et a!. The platelet membrane glycoprotein lib/IlIa complex. Structure, function, and relationship to adhesive protein receptors in nucleated cells. Ann N Y Acad Sci 1987; 509: 177

51 Bombeli T, Schwartz BR, Harlan JM. Endothelial cells undergoing apoptosis become proadhesive for nonactivated platelets. Blood 1999; 93: 3831

52 Pierson RN II! and Parker RE. Thromboxane mediates pulmonary vasoconstriction and contributes to cytotoxicity in pig lungs perfused with fresh human blood. Transplant Proc 1996; 28: 625

53 Horton R, Nadler J, Manogian C, et al. Prostacyclin is a key mediator of the vasodilator action of dopamine in humans. Adv Prostaglandin Thromboxane Leukot Res 1987; 17: 753

54 Haraldson A, Kieler-Jensen N, Nathorst-Westfelt U, et al. Comparison of inhaled nitric oxide and inhaled aerosolized prostacyclin in the evaluation of heart transplant candidates with elevated pulmonary vascular resistance. Chest 1998; 114: 780

55 Rolland PH, Bory M, Leca F, et al. Evidence for isosorbide dinitrate (ISDN) promoting effect on prostacyclin release by the lung and prostacyclin implication in ISDN-induced inhibition of pi ate let aggregation in humans. Prostaglandins Leukot Med 1984; 16: 333

56 Shapira OM, Rene H, Lider 0, et al. Prolongation ofrat skin and cardiac allograft survival by low molecular weight heparin. J Surg Res 1999; 85: 83

57 Dalmasso AP and Platt JL. Potentiation ofCl inhibitor plus heparin in prevention ofcomp!ementmediated activation of endothelial cells in a model of xenograft hyperacute rejection. Transplant Proc 1994; 26: 1246

58 Miller MJ and Sandoval M. Nitric Oxide. Ill. A molecular prelude to intestinal inflammation. Am J Physiol. 1999; 276: G795

59 Okayama N, Ichikawa H, Coe L, et al. Exogenous NO enhances hydrogen peroxide-mediated neutrophil adherence to cultured endothelial cells. Am J Physiol1998; 274: L820

60 Fiane AE, Videm V, Mollnes TE, et al. Inhibition of platelet aggregation by the GPilb/IIIa antagonist reopro does not significantly prolong xenograft survival in an ex vivo model. Transplant Int 1999; 12: 323

61 [mai M, Kaczmarek E, Koziak K, et al. Suppression of ATP diphosphohydrolase/C039 in human vascular endothelial cells. Biochemistry 1999; 38: 13473

199


200

v Discussiou

9 General discussion

Section V - Chapter 9

204

General discussion

GENERAL DISCUSSION

The potential clinical implementation of xenotransplantation, or the transplantation of

tissues and/or organs between species, has received considerable attention from both

governmental institutions and media in numerous countries over the last few years.

Renewed interest in xenotransplantation research has been fuelled by an increasing

shortage of donor organs and mortality of patients awaiting a donor organ. The

measures that have been undertaken to increase the donor-pool have thusfar not been

successful, and alternatives to xenotransplantation - i.e. the use of bioartificial organs.

stem cells, or gene therapy for organ failure - are still in early development.

Most investigators believe that, if xenotransplantation is to become a clinical reality,

the pig will be the most suitable source of organs for a number of reasons (1).

However, xenografts between widely disparate species i.e. discordant xenografis,

such as pig-to-primate, are rapidly rejected within minutes to hours by a humoraL

complement-dependent, mechanism, known as hyperacute rejection. Once hyperacute

rejection is averted, the rejection responses that follow are also thought to be

antibody-mediated, but most likely complement-independent, and may involve

cellular mechanisms (2). Although much progress has been made in overcoming some

of the immunological hurdles associated with pig-to-nonhuman primate

xenotransplantation, porcine organs do not routinely survive beyond three months in

nonhuman primates (3). As current evidence suggests that suppression of both the

humoral and cellular response to a discordant xenograft will require extremely intensive

immunosuppressive therapy, with its associated increased risks, it seems likely that

xenotransplantation will only prove successful if immunologic tolerance to the

transplanted pig organs can be achieved (4).

This dissertation describes our attempts at inducing immunological tolerance in the

pig-to-baboon xenotransplantation model through mixed chimerism. The condition

regimen we used was modified from others that have proven successful in various

models of allotransplantation and concordant xenotransplantation. The importance of

preformed, xenoreactive antibodies in these experiments is emphasized, and several

205


methods of depleting, or suppressing the production of, these antibodies are discussed

in detail. Furthermore, thrombotic complications were observed in baboons that

received porcine cells or organs. Additional experiments were designed to address

these complications; these are also included in this dissertation.

An introduction to xenotransplantation is presented in Chapter 1. The needs for

xenotransplantation, as well as the current understanding of the rejection responses

and potential modulation of these responses are discussed in detaiL From this chapter

we conclude that the discussed therapeutic modalities alone will not be able to

achieve long tenn, clinically relevant survival of xenografts. In this context, the

phenomenon of accomodation deserves some more attention. Although this state has

been described in sensitized humans receiving kidney allotransplants (5), as well as in

various small animal xenotransplantation combinations (6), it has to date not been

described in a pig-to-nonhuman primate xenotransplantation modeL In fact, with our

conditioning regimen aimed at inducing immunological tolerance, we have created an

environment that theoretically would be ideal to induce a state of accommodation.

The baboon recipients in these experiments are temporarily depleted of anti-Gal

antibodies and of terminal complement components when a porcine kidney is

transplanted. The returning anti-Gal antibodies, especially IgM, however, remain

capable of mediating graft rejection (Buhler et a!., submitted for publication). It

appears that the porcine endothelial cells, rather than becoming accommodated,

become activated by the induced antibody response and that accommodation may not

be achieved in pig-to-primate xenotransplantation. We therefore believe that

combinations of current therapies, further refinement of these therapies, and new

technologies designed to prevent or treat rejection (especially those aimed at inducing

immunological tolerance), need to be explored and developed.

Our protocol aimed at achieving mixed hematopoietic chimerism and thereby

inducing immunological tolerance in the pig-to-baboon model is presented in

Chapter 2. This protocol consists of induction therapy with splenectomy, whole body

irradiation, thymic irradiation, anti-thymocyte globulin, and multiple extracorporeal

immunoadsorptions of anti-Gal antibodies. The maintenance therapy consists of

206

General discussion

treatment with anti-CD154 mAb +/- cyclosporine, mycophenolate mofetil, and cobra

venom factor, after which a large number of grmvth factor-mobilized porcine leukocytes

(1 x 10 10 cellslkg) are infused into recipient baboons on each of 3 consecutive days (total

3 x 1010 cellslkg). In this chapter we present, for the first time in a large animal model,

successful depletion of baboon macrophages using medronate liposomes. The distinct

dimensions and properties of these liposomes render them susceptible to phagocytosis

by macrophages. Once phagocytosed, medronate interferes with the cell's metabolism

and causes cell death. When macrophage depletion is added to our conditioning

regimen for tolerance induction, significantly higher levels of porcine chimerism are

achieved in the recipient baboons, underlining the importance of recipient

macrophages in the clearance of donor porcine hematopoietic cells and the high

potential of medronate liposomes for other experimental xenotransplantation

protocols.

In addition, observations from other experiments suggest that an absence of anti-Gal

IgG may also be an important factor in allowing the development of chimerism, as

these antibodies are able to destroy target cells by antibody-dependent cell-mediated

cytotoxicity, independent of complement. Although anti-Gal antibodies are effectively

depleted from circulation by multiple extracorporeal immunoadsorptions, the return of

anti-Gal antibodies cannot be prevented, thereby reducing the possibility of achieving

mixed hematopoietic chimerism and immunological tolerance. The importance of

anti-Gal antibodies in mixed chimerism protocols and vascularized organ

xenotransplantation, as well as current methods for depleting, or suppressing the

function of these antibodies are described in Chapter 3. We conclude, based on a

comprehensive review of the literature, as well as on our own experience, that,

although anti-Gal antibodies may be successfully but temporarily depleted by

extracorporeal immunoadsorption, none of the immunosuppressive agents currently

employed are significantly successful at preventing their return following depletion.

However, the ability of anti-CD 154 mAb to prevent the induced antibody response to

pig cells does represent a significant step in overcoming the immunologic barriers to

xenotransplantation, and will undoubtedly facilitate the survival of transplanted pig

organs in primates and the induction of tolerance.

207

Section V·- Chapler 9

Some of the future studies described in chapter 3 with respect to the development of

specific anti-B cell or anti-plasma cell antibodies in an effort to prevent the

production of anti-Gal antibodies are explored in Chapter 4. In extensive in vivo and

in vitro experiments, we demonstrated that baboon B cells can be efficiently depleted

with whole body irradiation or with specific anti-B cell mAbs or irnmunotoxins.

Additionally, combining irradiation with a rnAb directed against the B cell-specific

CD20 determinant led to depletion of B cells for > 3 months. However, this does not

lead to a clinically significant reduction in the rate or extent of return of anti-Gal

antibodies following immunoadsorption. These observations suggest that B cells are

not the major source of anti-Gal antibody production in baboons, and that attention

must be directed towards suppressing plasma cell and B cell blast function. This was

confirmed by our findings that the major secretors of anti-Gal antibodies in baboons

are CD38-positive cells, or plasma cells. Unfortunately, the anti-CD38 irnrnunotoxin

we studied was not effective in vivo, potentially due to various reasons, including

suboptimal conjugation of the rnAb to the toxin.

The next chapters focus on the thrombotic complications that are associated with pig

to-baboon xenotransplantation. There are historical precedents for the development of

coagulation abnormalities and thrombocytopenia in association with solid organ

xenograft rejection, and these data are confirmed by the studies described in Chapter

5. In this chapter, we also demonstrate that the infusion of porcine hematopoietic cells

is associated with vvidespread thrombotic vascular injury with deleterious

consequences for the recipient. The mechanisms underlying the thrombotic

complications in these settings are still unclear. It is possible that varying levels of

immune mediators within the vascularized xenograft could promote vascular

thrombosis, as a component of the inflammatory response ab initio with endothelial

cell activation. With respect to porcine hematopoietic cell transplantation, we

hypothesized that these cells directly induce platelet aggregation and subsequent

sequestration of platelets in the microvasculature of recipient baboons, or that the

conditioning regimen aimed at inducing mixed chimerism contains components that

may initiate platelet aggregation and thrombocytopenia.

208

General discussion

In Chapter 6 we demonstrate that porcine hematopoietic cells mediate aggregation of

baboon platelets. Furthermore, we present evidence that eptifibatide, a substance that

prevents the GPIIb/lIIa platelet-receptor from binding to fibrinogen, thereby

precluding crosslinking of platelets, can fully abrogate platelet aggregation induced

by these cells in vitro. Purification of the precursors from porcine PBPC and/or

treatment of baboons with eptifibatide may be beneficial in preventing these sequelae.

These findings are confirmed in Chapter 7 where, in addition, each of the individual

components of the conditioning regimen are assessed for their ability to induce

platelet aggregation in baboons. Interestingly, none of the components of the

conditioning regimen are found to be pro-aggregatory, but cyclophosphamide appears

to have protective, anti-aggregatory properties and may therefore provide an

alternative strategy to whole body irradiation in our attempts to achieve mixed

hematopoietic chimerism and the induction of tolerance.

Finally, in Chapter 8 other pharmacologic agents aimed at preventing platelet

aggregation III baboons are examined. We demonstrate that, although

aurintricarboxylic acid and fucoidin are potent inhibitors of platelet aggregation, the

experimental use of these agents in baboons may be prohibited by an associated high

risk of bleeding. Heparin leads to good inhibition of thrombin-induced platelet

aggregation at doses that cause only minor prolongation of the partial thromboplastin

time, allowing for its use in xenotransplantation models. Eptifibatide leads to

excellent inhibition of platelet aggregation without any untoward side effects and is

therefore a good candidate to be incorporated into Oill future pig-to-nonhuman

primate studies.

The successful induction of stable immunological tolerance in pig-to-baboon

xenotransplantation will, beyond any doubt, pave the way to clinical

xenotransplantation. The above discussion deady points out that many factors, both

immunological and not, stand in the way of mixed chimerism and tolerance. Based on

the previous studies, it has become evident that anti-Gal antibodies remain a major

209


problem in xenotransplantation. Recent experiments perfonned at our own laboratory

(not included in this dissertation) with in vivo depletion of anti-Gal antibodies by

infusion of bovine serum albumin conjugated to Gal (BSA-Gal) to naIve baboons,

baboons undergoing our tolerance induction protocol, or baboons receiving porcine

kidney xenografts (in conjunction with extracorporeal immunoadsorption and

costimulatory blockade with an anti-CD154 mAb), demonstrated complete or near

complete depletion of anti-Gal antibodies sustained for periods of up to 30 days

(Teranishi and Gollackner, unpublished data). The return of anti-Gal antibodies

following cessation of the BSA-Gal was more delayed than observed in controls that

did not receive BSA-Gal. In addition to neutralizing anti-Gal antibodies, this agent

may, therefore, possibly tolerize anti-Gal antibody-producing cells by binding to their

antigen receptors. We therefore believe that future studies must focus on the

refinement of BSA-Gal, as well as on the development of agents that prevent the

production of anti-Gal antibodies, such as efficient anti-plasma cell immunotoxins.

Additionally, the potential of macrophage depletion in pig-to-nonhuman primate

tolerance induction needs to be further explored and possibly implemented in refined

protocols, Furthennore, the etiology of the associated thrombotic complications must

be elucidated.

In conclusion, this dissertation discusses some of the major immunological hurdles

that preclude clinical implementation of xenotransplantation, and offers insight into

current attempts at inducing immunological tolerance through mixed hematopoietic

chimerism. The problem of anti-pig antibodies is addressed, as well as associated

thrombotic disorders, and current and novel therapeutic modalities are presented.

Based on the results presented in this dissertation, the addition of a specific and

effective anti-plasma cell immunotoxin, macrophage depletion with medronate

liposomes, and prevention of platelet aggregation with eptifibatide, to the standard

conditioning regimen as discussed in Chapter 2, may facilitate the induction of mixed

chimerism and immunological tolerance. Further refinement of BSA-Gal, to achieve

in vivo depletion of anti-Gal antibodies, as well as using composite thymo-organs for

T cell tolerance may also be beneficial. The author hopes that the experiments and

210

General discussion

findings described will be of benefit to the future of xenotransplantation, and that this

exciting field will eventually become a part of clinical medicine.

REFERENCES

1 Sachs DH. The pig as a potential xenograft donor. Vet Imm Immunopath 1994; 43: 185

2 Bach FH .. Robson SC, Winkler H, et al. Barriers to xenotransplantation. Nat Med 1995; I: 869

3 Zaidi A, Schmoeckel M, Bhatti F, et al. Life-supporting pig-to-primate renal xenotransplantation using genetically modified donors. Transplantation 1998: 65: 1584

4 Wekerle T, Sykes M. Mixed chimerism as an approach for the induction of transplantation tolerance. Transplantation 1999; 68: 459

5 Alexandre GP J, Squifflet JP, De Bruyere M, et a!. Present experiences in a series of26 ABOincompatible living donor renal allografts. Transplant Proc J 987; 19: 4538

6 Bach FH, Ferran C, Hechenleitner P, et aL Accomodation of vascularized xenografts: expression of "protective genes" by donor endothelial cells in a host Th2 cytokine environment. NatMed 1997; 3: 196

211

Section V - Chapler 9

212

10 Summary in Dutch

Section V-Chapter 10

214

Summwy in Dutch

D1SCUSSIE

Het begrip xenotransplantatie, ofwel het transplanteren van weefsel eulof organen

tussen diersoorten, is de laatste jaren onderwerp van discussie geweest op nationaal en

internationaal niveau, zowel bij overheidsinstanties als in de publieke media.

Vanwege het steeds groter wordend tekort aan orgaandonoren, met daarbij een

stijgende mortaliteit van patienten met tenninaal orgaanfalen die op de wachtlijst

staan voer een donororgaan, is hemieuwde belangstelling voor xenotransplantatie

onderzoek ontstaan. De huidige maatregelen welke getroffen zijn am het aantal

donoren te vergroten schieten helaas tekort, en altematieven voor transplantatie -

zoals het gebruik van bioartificiele organen, stamcellen, of gentherapie bij

orgaanfalen - zijn nog niet voldoende ontwikkeld.

Het varken wordt door veel wetenschappers gezien als de ideale leverancier voor

donororganen (1). Het transplanteren van organen tussen diersoorten die fylogenetisch

ver van elkaar zijn verwijderd, zoals van het varken naar de primaat, gaat echter

gepaard met een zeer snelle en heftige afstotingsreactie. Deze afstoting, ook wei

hyperacute afstoting genoernd, is antilichaam-gemedieerd en cornplement-afhankelijk,

en treedt binnen enkele minuten tot uren na transplantatie op. Indien het lukt deze

afstotingsvorm af te wenden, bijvoorbeeld door antilichaam of complement depletie

of door varkens te gebruiken die transgeen zijn voor humaan complement regulerende

eiwitten, treden andere afstotingsverschijnselen op. Deze zijn eveneens antilichaam

gernedieerd maar waarschijnlijk cornplement-onafuankelijk, en bevatten daarnaast

cellulaire cornponenten (2). Hoewel veel vooruitgang is geboekt in het begrijpen en

behandelen van sommige van deze immunologische barrieres, lukt het niet

routinernatig overleving van meer dan drie maanden te verkrijgen van varkensorganen

in primaten (3). De huidige bewijslast suggereert dat de humorale en cellulaire

afstotingsresponsen tegen xenotransplantaten in het varken-naar-prirnaat model

dennate hevig zullen zijn dat deze aileen te behandelen zullen zijn met bijzonder hoge

doseringen immuunsuppressiva, hetgeen gepaard zal gaan met onaanvaardbaar hoge

risico's op infecties en rnaligniteiten. Het verkrijgen van immunologische tolerantie in

dit model, waarbij een specifieke hyporesponsiviteit voor varkensantigenen in de

215


primaat-ontvanger optreedt, is derhalve essentieel voordat xenotransplantatie klinisch

gelmp1ementeerd zal worden (4).

Dit proefschrift beschrijft onze pogingen immunologische tolerantie te induceren in

het varken-naar-baviaan xenotransplantatie model via het verkrijgen van gemengd

hematopoietisch chimerisme. Het gebruikte conditioneringsprotocol is gemodelleerd

naar succesvolle protocollen binnen allotransplantatie en concordante

xenotransplantatie. Het belang van voorgevormde, xenoreactieve antilichamen in deze

experimenten wordt benadrukt en diverse methoden van depleteren of onderdrukken

van de produktie van deze antilichamen worden in detail besproken. Hiemaast worden

de thrombotische complicaties welke ZIJn gesignaleerd In bavianen die

varkensorganen of hematopoietische cellen ontvangen gepresenteerd. T evens worden

de aanvullende experimenten die zijn uitgevoerd om deze complicaties te analyseren

en beperken beschreven.

Een introductie tot xenotransplantatie wordt gegeven in Hoofdstuk 1. Het belang en

de noodzaak van deze vorm van transplantatie, alsmede het huidig begrip en de

behandelingsmogelijkbeden van de afstotingsverschijnselen, worden in detail

besproken. Wij concluderen uit dit hoofdstuk dat de huidige therapie op zichzelf

ontoereikend is om langdurige, klinisch relevante overleving van xenotransplantaten

te verkrijgen. In dit verband verdient het begrip accommodatie enige toelichting. Het

houdt in dat de getransplanteerde organen overleven ondanks de aanwezigheid van

antilichamen gericht tegen antigenen van deze transplantaten. Accommodatie is

beschreven bij gesensitiseerde patienten die een niertransplantatie ondergaan (5)

alsmede in verscheidene kleine proefdiermodellen van xenotransplantatie (6). Tot

heden is dit fenomeen echter niet aangetoond in het varken-naar-primaat

xenotransplantatie model. In het door ons bestudeerde tolerantie-inductie protocol

wordt een cmgeving gecreeerd welke theoretisch ideaal zou zijn voor het optreden

van accommodatie. Irnmers, de bavianen in deze experimenten zijn gedepleteerd van

anti-Gal antilichamen en terminale complement componenten op het moment dat een

varkensnier wordt getransplanteerd. De anti-Gal antilichamen welke terugkeren na

depletie, in het bijzonder IgM antilichamen, blijven echter in staat afstoting te

216

Summary in Dutch

veroorzaken (Buhler et al., ingestuurd v~~r publikatie). Kennelijk ondergaan de

varkensendotheelcellen in plaats van accommodatie, activatie door de geYnduceerde

antilichaamrespons in dit model en is het mogelijk dat accommodatie niet te

verkrijgen zal zijn in varken-naar-primaat xenotransplantatie. Wij denken derhalve dat

wellicht combinaties van therapieen, verdere verfijning van bestaande behandelingen

en het ontwikkelen van nieuwe strategieen gericht op het voork6men danwel

behandelen van afstoting dienen te worden ontwikkeld. In het bijzonder dient de

inductie van immW1oiogische tolerantie aandacht te krijgen.

Het protocol gericht op het verkrijgen van gemengd hematopoietisch chimerisme met

hierbij de inductie van immunologische tolerantie in het varken-naar-baviaan model

wordt gepresenteerd in Hoofdstuk 2. Dit protocol bestaat uit inductietherapie van de

baviaan met behulp van splenectomie, totale lichaams- en thymus bestraling, het

toedienen van anti-thymocyten globuline, en multipele extracorporele

immunoadsorpties van anti-Gal antilicharnen. De onderhoudsbehandeling bestaat uit

het toedienen van anti-CD154 monoklonale antilichamen +/- cyclosporine,

mycofenolaat mofetil en cobra gif, waama verspreid over drie dagen een grote

hoeveelheid groeifactor-gernobiliscerde varkens lcukocyten (totaal 3 x 1010 cellenlkg)

wordt toegediend aan de baviaan. Hier presenteren wij voor de eerste keer in een grote

proefdieren model succesvolle depletie van bavianen macrofagen met behulp van

medronaatliposomen. Deze liposomen hebben specifieke kenmerken en afrnetingen

welke ze ontvankelijk rnaken voor fagocytose door macrofagen. Het gefagocyteerde

medronaat verstoort vervolgens het rnetabolisme van de cel en leidt tot celdood.

Wanneer macrofaagdepletie wordt toegevoegd aan het basis-conditionerings protocol

zoals hierboven uiteengezet worden beduidend hogere waarden van varkenschimerisrne

in de ontvangende bavianen aangetoond. Hiennee wordt het belang van macrofagen in

het wegvangen van varkenscellen uit de circulatie van bavianen onderstreept en lijkt het

interessant macrofaagdepletie ook in andere xenotransplantatiemodellen te bestuderen.

Belangrijk voor het verkrijgen van gemengd hematopoietisch chimerisme is de

afwezigheid van anti-Gal antilicharnen, in het bijzonder anti-Gal IgG. Uit eerdere

studies is gebleken dat deze antilichamen cellen kunnen destrueren middels

217


antilichaam-afhankelijke cel-gemedieerde cytotoxiciteit, onatbankelijk van

complement. Anti~Gal antilichamen kunnen effectief worden verwijderd uit de

circulatie middels achtereenvolgende extracorporele immunoadsorpties. De terugkeer

van anti-Gal antilichamen wordt hiennee echter niet voorkomen waardoor de kans op

het verkijgen van chimerisme en irnmunologische tolerantie wordt venninderd. Het

belang van anti-Gal antilichamen in gemengd-chimerisme protocollen en

xenotransplantatie van gevasculariseerde organen, alsmede huidige

behandelingsmethoden voor het depieteren of venninderen van de functie van deze

antilichamen worden beschreven in Hoofdstuk 3. Wij concluderen, gebaseerd op de

meest recente literatuur en onze eigen ervaring dat, hoewel tijdelijke depietie van anti

Gal antilichamen zeer goed mogelijk is middels extracorporele irnmunoadsorptie, niet

een van de gangbare immmllsuppressiva de terugkeer van antilichamen na depletie

kan voorkomen. Het vennogen van het anti-CD 154 antilichaam om de gei'nduceerde

antilichaamrespons tegen varkenscellen te voorkomen is echter weI een significante

bevinding en zal zeker de overleving van varkensorganen in primaten verbeteren en

de inductie van tolerantie vergemakkelijken.

Sommige van de in hoofdstuk 3 beschreven vervolgstudies met betrekking tot het

ontwikkelen van specifieke anti-B eel of anti-plasmacel antilichamen met als doel

anti~Gal antilichaamproduktie te voorkomen, worden bestudeerd in Hoofdstuk 4. In

uitgebreide in vivo en in vitro experimenten demonstreren wij dat B cellen in

bavianen efficient kunnen worden gedepleteerd middels totale lichaamsbestraling of

door toediening van specifieke anti-B eel monoclonale antilichamen of

immunotoxines. De combinatie van bestraling met een monoclonaal antilichaam

gericht tegen de B cel-specifieke detenninant CD20 resulteert in depletie van B cellen

voor meer dan 3 maanden. Dit leidt echter niet tot vennindering van anti~Gal

antilichaamproduktie. Deze observaties suggereren dat B cellen niet de voomaamst

verantwoordelijke cellen zijn voor antilichaam produktie in bavianen en dat aandacht

dient te worden besteed aan het onderdrukken van de [unktie van plasmacellen. Dit

blijkt ook uit in vitro experimenten waarin het merendeel van anti-Gal antilichaam

secreterende cellen CD38-positief zijn, een marker voor plasmacellen. Helaas was het

anti-CD38 immunotoxine dat wij hanteerden in onze in vivo experimenten niet

218

Summary in Dutch

effectief, onder andere vanwege een suboptimale conjugatie van het antilichaam met

het toxine.

In de volgende hoofdstukken wordt aandacht besteed aan de thrombotische

complicaties die geassocieerd worden met varken-naar-baviaan xenotransplantatie.

Het ontstaan van stollingsstoomissen en thrombocytopenie samenhangend met de

xenotransplantatie van gevasculariseerde organen is in het verleden reeds beschreven

en wordt door ooze onderzoeksresultaten bevestigd in Hoofdstuk 5. In dit hoofdstuk

demonstreren wij ook dat het transplanteren van varkenshematopoietische cellen

geassocieerd is met indrukwekkende thrombotische vasculaire schade met schadelijke

gevolgen voor de ontvanger. Het hieraan ten grondslag liggend mechanisme is

onduidelijk. Mogelijk spelen immuunmediatoren binnen het xenotransplantaat een rol.

Deze zouden bijvoorbeeld thrombose kunnen bevorderen als onderdeel van een

inflarnmatoire reactie welke optreedt na endotheelcelactivatie. In het geval van

hematopoietische celtransplantatie IS het mogelijk dat deze cellen direct

bloedplaatjesaggregatie veroorzaken met een hieropvolgende sequestratie van

bloedplaatjes in de microcirculatie van de baviaan ontvangers. Ook kunnen

individuele componenten van het conditioneringsregime bloedplaatjesaggregatie met

thrombocytopenie veroorzaken.

In Hoofdstuk 6 tonen wij dat varkenshematopoietische cellen aggregatie kunnen

medieren van bavianenbloedplaa~es. Tevens presenteren Vvij dat eptifibatide, een

GPIIb/IIIa receptor antagonist, deze aggregatie in vitro volledig kan voorkomen. Het

venvijderen van debris uit het varkenshematopoietische stamcellen produkt en/of het

behandelen van bavianen met eptifibatide kan helpen deze complicaties te

voorkomen.

Deze bevindingen worden nogmaals bevestigd in Hoofdstuk 7. T evens worden de

individuele componenten van het conditioneringsregime beoordeeld op hun vermogen

thrombocyten aggregatie te induceren in bavianen. Naast het feit dat geen van de

componenten inductie van aggregatie veroorzaakt, wordt aangetoond dat

cyclofosfamide beschermende, anti-aggregatoire eigenschappen bezit. Dit middel kan

219


derhalve in onze pogingen gemengd hematopoietisch chimerisme te bereiken een

alternatief betekenen voor totale Iichaamsbestraling.

Tenslotte worden in Hoofdstuk 8 andere fannacologische middelen getest op hun

vennogen thrombocytenaggregatie te remmen in bavianen. Middelen als

aurintricarboxylzuur en fucoidin blijken krachtige remmers van aggregatie te zijn. Het

gebruik hiervan wordt echter verhinderd door een onaanvaardbaar hoog risico op

bloedingscomplicaties. Heparine is een bekende remmer van thrombine-geinduceerde

bloedplaatjesaggregatie en kan worden gebruikt in doseringen die slechts een kleine

verlenging van geactiveerde partiele thromboplastinetijd veroorzaken. Eptifibatide

leidt tot een zeer goede inhibitie van thrombocytenaggregatie zonder duidelijke

bijwerkingen en is hierdoor een goede kandidaat om te gebruiken binnen onze

toekomstige xenotransplantatieprotocollen.

De succesvolle inductie van stabiele immunologische tolerantie in varken-naar

baviaan xenotransplantatie zal ongetwijfeld de voorv.raarde scheppen voor klinische

xenotransplantatie. Het bovenstaande illustreert dat vele factoren, zowel

immunologische als andere, in de weg staan van gemengd chimerisme en tolerantie

voor varkensorganen. Gebaseerd op voorgaande studies is duidelijk geworden dat

anti-Gal antilichamen een groot probleem vonnen in xenotransplantatie. Wij hebben

recent experimenten uitgevoerd (niet beschreven in dit proefschrift) om in vivo

depletie van anti-Gal antilichamen te verkrijgen in naIve bavianen, in bavianen die

ons tolerantie-inductieprotocol ondergaan en In bavianen die

varkensniertransplantaten ontvangen, door albumine van kalveren geconjugeerd met

Gal (BSA-Gal) toe te dienen. Met dit middel wordt (bijna) complete depletie van anti

Gal antiliehamen verkregen tot 30 dagen (Teranishi and Gollaekner, ruet

gepubliceerde data) na toediening. De terugkeer van anti-Gal antilichamen, nadat

behandeling met BSA-Gal is gestaakt, verloopt vervolgens langzarner dan bij

bavianen die niet met BSA-Gal worden behandeld. Naast het vermogen van BSA-Gal

om anti-Gal antilichamen te neutralizeren, kan het waarschijnlijk ook anti-Gal

antilichaamproducerende cellen tolerizeren door hun antigeenreceptoren te binden.

Wij vinden derhalve dat, naast de al eerder genoemde studies gericht op het

220

Summary in Dutch

ontwikkelen van middelen die de produktie van anti-Gal antilichamen kunnen

remmen. zoals efficiente anti-plasmacel immunotoxinen, toekomstige studies gericht

moeten zijn op het verfljnen van het gebruik van BSA-GaL

Tevens dient het potentieel van macrofaagdepletie in varken-naar-primaat tolerantie

inductie modellen nader onderzocht te worden en zo mogelijk gelmplementeerd te

worden in verbeterde protocollen. Ook zal de etiologie van het v66rkomen van

thrombotische complicaties in deze modellen bestudeerd moeten worden.

Sarnenvattend kan worden gesteld dat in dit proefschrift een aantal van de

belangrijkste immunologische obstakels voor klinische impiementatie van

xenotransplantatie wordt besproken. T evens wordt inzicht gegeven in de huidige

mogelijkheden om immunologische tolerantie te verkijgen in het pre-klinische

varken-naar-baviaan gemengd-chimerisme model. Het probleem van anti-varkens

antilichamen wordt toegelicht, begeleidende thrombotische complicaties worden

beschreven en huidige en nieuwe behandelingsmogelijkheden hiervan worden

voorgesteld. Op grond van de resultaten beschreven in dit proefschrift zal een

protocol dat, naast de componenten van het conditioneringsregime zoals beschreven

in Hoofdstuk 2. tevens een specifieke en effectieve anti-plasmacel immunotoxine,

medronaatliposomen v~~r macrofaagdepletie, alsmede eptifibatide ter voorkoming

van thrombocytenaggregatie, bevat, moge1ijk de inductie van gemengd chimerisme en

immunologische tolerantie faciliteren. De auteur hoopt dat de experimenten en

resultaten welke beschreven worden in dit proefschrift, van belang zijn voor de

toekomst van xenotransplantatie en dat dit boeiende vakgebied deel zal gaan uitmaken

van de tmnsplantatiegeneeskunde.

REFERENCES

I Sachs DH. The pig as a potentia! xenograft donor. Vet Imm [mmunopath ! 994: 4),185

2 Bach FH, Robson SC, Winkler H, et al. Barriers to xenotransplantation. Nat Med 1995; 1,869

3 Zaidi A. Schmoeckei M, Bhatti F, et aL Life-supporting pig-to-primate renal xenotranspiantation using genetically modified donors. Transplantation \998; 65: 1584

221


4 Wekerle T, Sykes M. Mixed chimerism as an approach for the induction of transplantation tolerance. Transplantation 1999; 68: 459

5 Alexandre GPJ, Squifflet JP, De Bruyere M, et al. Present experiences in a series of26 ABOincompatible living donor renal allografts. Transplant Proc 1987; 19: 4538

6 Bach FH, Ferran C, Hechenleitner P, et al. Accomodation of vascularized xenografts: expression of "protective genes" by donor endothelial cells in a host Th2 cytokine environment. Nat Med 1997; 3: 196

222

LIST OF ABBREVIATIONS

I-BI

3H-Leu

aGal

o::CD20mAb

o::CD22-IT

o::CD2S-lT

ocCD38 mAb

ocCD38-lT

ADP

Anti-aGal Ab:

ATA

ATG

AVR

BM

cpp

CVF

dg

DIC

EFT

EIA

FDP

GP

HAR

HBSS

IT

1. v.

IVIg

LDH

LN

I-benzylimidazole

3H-Leucine

galal-3Gal epitope

anti-CD20 monoclonal antibody Rituximab

anti-CD22-ricin A immunotoxin

anti-CD25-ricin A irnmunotoxin

anti-CD38 monoclonal antibody OKTI 0

anti-CD38-ricin A immunotoxin

adenosine diphosphate

anti-Gala 1-3Gal antibodies

aillintricarboxylic acid

antithyrnocyie globulin

acute vascular rejection

bone marrow

cyclophosphamide

cobra venom factor

deglycosylated

disseminated intravascular coagulation

eptifibatide

extracorporeal immunoadsorption

fibrinogen degradation products

glycoprotein

hyperacute rejection

Hank's balanced salt solution

irnmunotoxin

intravenous

intravenous immunoglobulin

lactate dehydrogenase

lymph node

223

mAb

ML

MPS

NA

NMCR

NO

NPN

PCIx

PGJ,

pIL3

POIx

pPBPC

PRP

pSCF

PI

PTI

ricin A

SFU

IM

TIP

vWF

WBI

224

monoclonal antibody

medronate liposomes

mononuclear phagocyte system

nicotinamide

non-myeloablative conditioning regimen

nitric oxide

nitroprusside sodium

pig cell transplantation

prostacyclin

porcine interleukin 3

pig organ (kidney or heart) transplantation

porcine hematopoietic progenitor cells

baboon platelet-rich plasma

porcine stem cell factor

prothrombin time

partial thromboplastin time

RIA

spot forming units

thrombotic microangiopathy

thrombotic thrombocytopenic purpura (rnicroangiopathy)

von Willebrand factor

whole body irradiation

LIST OF PUBLICATIONS

Publications related to this dissertation:

Alwayn IPJ, Buhler L, Basker M, and Cooper DKC. Immunomodulation strategies in

xenotransplantation. In: Current and Future Immunosuppressive therapies following

Transplantation. Sayegh M and Remuzzi G (Eds.). In press.

Alwayn IP J, Basker M, Buhler L, Harper D, Abraham S, Kruger Gray H, A "wad M,

Down J, Rieben R, White-Scharf ME, Sachs DH, Thall A, and Cooper DKC. Clearance

of mobilized porcine peripheral blood progenitor cells is delayed by depletion of the

phagocytic reticuloendothelial system in baboons. Transplantation 2001. In press.

Alwayn IPJ, Basker M, Buhler L, and Cooper DKC. The problem of anti-pig antibodies

in pig-to-primate xenografting: current and novel methods of depletion and/or

suppression of production of anti-pig antibodies. Xenotransplantation 1999;6(3):157-

168.

Basker M, Alwayn IPJ, Treter S, Harper D, Buhler L, Andrews D, Thall A, Lambrigts

D, Awwad M, White-Scharf M, Sachs DH, and Cooper DKC. Effects of B cell/plasma

cell depletion or suppression on anti-Gal antibody in the baboon. Transpl. Proc.

2000;32(5).'1009.

Alwayn IPJ, Xu Y, Basker M, Wu C, Buhler L, Lambrigts D, Treter S, Harper D.

Kitamura H, Vitetta E, Awwad M, White-Scharf ME, Sachs DH, Thall A, and Cooper

DKC. Effects of specific anti-B and/or anti-plasma cell immunotherapy on antibody

production in baboons: depletion of CD20- and CD22-positive B cells does not result in

significantly decreased production of anti-cxGal antibody. Xenotransplantatiol1 2001. In

press.

225

AlwaYD IPJ, Buhler L, Basker M, Goepfert C, Kawai T, Kozlowski T, leriDo F, Sachs

DH, Sackstein R, Robson SC, and Cooper DKC. Coagulation I Thrombotic disorders

associated with organ and cell xenotransplantation. Transp/. Froc. 2000;32(5):1099.

Buhler L, Basker M, Alwayn IPJ, Goepfert C, Kitamura H, Kawai T, Gojo S, Kozlowski

T, lerino FL, Awwad M, Sachs DH, Sackstein R, Robson SC, and Cooper DKC.

Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell

transplantation in nonhuman primates. Transplantation 2000;70(9):1323-1331.

AlwaYD IPJ, Buhler L, Appel JZ Ill, Goepfert C, Eva Csizmadia, Hiroshi Kitamura,

Sackstein R, Cooper DKC, and Robson Sc. Mechanisms of thrombocytopenia following

xenogeneic hematopoietic progenitor cell transplantation. Transplantation 2001. In press.

AlwaYD IPJ, Appel JZ Ill, Buhler L, DeAngelis HA, Cooper DKC, and Robson SC.

Modulation of platelet aggregation in baboons: implications for mixed chimerism in

xenotransplantation. I. The roles of individual components of a transplantation

conditioning regimen and of pig peripheral blood progenitor cells. Transplantation 2001.

In press.

Appel JZ Ill, AlwayD IPJ, Buhler L, Correa LE, Cooper DKC, and Robson SC.

Modulation of platelet aggregation in baboons: implications for mixed chimerism in

xenotransplantation. 2. The effects of cyclophosphamide on pig peripheral blood

progenitor cell-induced aggregation. Transplantation 2001. In press.

Alwayn IPJ, Appel JZ Ill, Goepfert C, Buhler L, Cooper DKC, and Robson SC.

Inhibition of platelet aggregation in baboons: therapeutic implications for

xenotranspJantation. Xenotransplantation 2000; 7(4):247-257.

226

Other publications:

Alwayn IP J, Xu R, Adler WH, and Kiltur DS, Does high MHC class II gene expression

in normal lungs account for the strong irnmunogenicity of lung allografts?

Transp/' 1nt, 1994,'7(1):43-46,

Scheringa M, Alwayn IPJ, and Marquet RL. Chronic rejection after xenotransplantation.

Xeno 1996,'4(1): 13,

Scheringa M, Alwayn IPJ, Bouwman E, IJzermans JNM, and van Bockel lH. Aorta

transplantation as a model to study hyperacute, acute, and chronic rejection of xenografts.

Xenotransp1antation 1996,'3 (3),'231-236,

Alwayn IPJ, van Bockel HJ, Daha MR, and Scheringa M. Hyperacute rejection in the

guinea pig-to-rat model without formation of the membrane attack complex. Transp!.

1mmuno/' 1999,-7(3).'177-182.

Basker M, Buhler L, AlwaYD IPJ, Appel JZ III, and Cooper DKC. Pharmacotherapeutic

agents in xenotransplantation. Exp. Opin. Pharmacother. 2000; 1(4):757-769.

Buhler L, Treter S, McMorrow I, Neethling FA, AlwayD I, A wwad M, Thall A, Cooper

OK, LeGuern C, Sachs DH. Injection of porcine anti-idiotypic antibodies to primate anti

Gal antibodies leads to active inhibition of serum cytotoxicity in a baboon. Transp!. Proc.

2000,32(5):1102.

Goepfert C, Buhler L, Wise R, Basker M, Gojo S, Imai M, AlwaYD I, Sachs DH,

Sackstein R, Cooper DK, Robson sc. Von willebrand factor concentration, multimeric

patterns, and cleaving protease activity in baboons undergoing xenogeneic peripheral

blood stem cell transplantation. Transpl. Froc. 2000;32(5):990.

227

Watts A, Foley A, Awwad lVI, Treter S, Oravec G, Buhler L, Alwayn IPJ, Kozlowski T,

Lambrigts D, Gojo S, Basker M, White-Scharf ME, Andrews D, Sachs DH, and Cooper

DKC. Plasma perfusion by apheresis through a Gal immunoaffinity column successfully

depletes anti-Gal antibody experience with 320 aphereses in baboons.

Xenotranspiantation 2000; 7(3): 181-185.

Buhler L, Basker M, Alwayn IPJ, and Cooper DKC. Therapeutic strategies for

xenotranspiantation. In: Xenotransplantation. Platt JL (Ed.). A}l1S Press, Washington,

DC.pp1l7-135.

Appel JZ Ill, Alwayn IPJ, and Cooper DKC. Xenotransplantation - the challenge to

current psychosocial attitudes. Progress in Transplantation 2000; 1 0:217.

Buhler L, Alwayn IPJ, Appel lZ III. Robson Sc. and Cooper DKC. Anti-CD154

monoclonal antibody and thromboembolism (letter). Transplantation 2001,'71:491.

Buhler L, Alwayn IPJ, et al. Pig hematopoietic cell chimerism in baboons conditioned

with a nonmyeloablative regimen and CDl54 blockade. Transplantation 2001. Inpress.

Alwayn IPJ, Buhler L, Teranishi K, Gollackner B, and Cooper DKC.

Immunosuppression for transgenic pig-to-nonhuman primate organ grafting. Current

Opinions in Transplantation 2001. In press.

Alwayn IP J and Robson Sc. Understanding and preventing the coagulation disorders

associated with xenograft rejection. Graft 2001. In press.

Alwayn IPJ, Basker M, and Cooper DKC. Suppressing natural antibody production.

Graft 2001. In press.

228

Buhler L, Goepfert C, Kitamura H, Alwayn IPJ, Basker M, Gojo S, Chang Q, Down J,

Tsai H, Sachs DH, Cooper DKC, Robson SC, and Sackstein R. Porcine hematopoietic

cell xenotransplantation in nonhuman primates is complicated by thrombotic

microangiopathy. Bone Marrow Transplantation 2001. In press.

Buhler L, Yamada K, Kitamura H, Alwayn IPJ, Basker M, Appel JZ m, Colvin RE,

White-Scharf ME, Sachs DH, Robson SC, Awwad M, and Cooper DKC. Pig kidney

transplantation in baboons: IgM is associated with acute humoral xenograft rejection and

disseminated intravascular coagulation. Submittedjor publication.

McMorrow 1M, Buhler L, Treter S, Neethling F, Alwayn IPJ, Comraek CA, Kitamura H,

Sachs DH, A'WWad M, DerSimonian H, Cooper DKC, and LeGuem C. Down-regulation

of the anti-Gal xenogeneic response in vivo through anti-idiotypic modulation. Submitted

for publication.

Buhler L, Alwayn IPJ, Basker M, Oravec G, Thall A, White-Scharf ME, Sachs DH,

A wwad M, and Cooper DKC. CD40-CD 154 pathway blockade requires host

macrophages to induce humoral unresponsiveness to pig hematopoietic cells in baboons.

Submitted/or publication.

Buhler L, Deng S, O'Neil J, Kitamura H, Koulmanda M, Alwayn IPJ, Appel JZ, Awwad

M, Sachs DH, Weir G, Squifflet JP, Cooper DKC, Morel P. Adult porcine islet

transplantation in baboons treated with a nonmyeloablative regimen and CD 154

blockade. Submitted for publication.

229

ACKNOWLEDGEMENTS

Many people have contributed to the realization of this dissertation. In particular, I would

like to acknowledge the support, motivation, inspiration, and friendship of the [oHowing

people:

Prof.dr. D.K.C. Cooper. prof.dr. D.H. Sachs. prof.dr. l. leekel, dr. J.N.M. IJzermans,

prof.dr. R. Benner, prof.dr. F. Grosveld, prof.dr. W. Weimar, the TBRC fellows, techs,

and staff, the RdGG surgical staff and residents, Patrick Vriens, Heio Stockmann, the

Alwayn family, and :)

231

CURRICULUM VITAE AUCTORIS

The author was born on September 30th, 1968 in Leidschendam, The Netherlands. After

graduating from high school at the Huygens Lyceum in Voorburg in 1986, he attended

medical school at the University of Leiden. During medical school, he worked as a

student assistant at the Thoracic Intensive Care Unit, Leiden University Medical Center

(prof.dr. H.A. Huysmans), perfonned research electives at the Laboratory for

Experimental Surgery, University Hospital Rotterdam 'Dijkzigt' (dr. R.L. Marquet) and

the Division of Transplantation Surgery, Johns Hopkins Hospital, Baltimore, U.S.A. Cdr.

D.S. Kittur), and performed a clinical subinternship at the GJ. Gold Service, Department

of Surgery, lohns Hopkins Hospital, Baltimore, U.S.A. (prof.dr. 1. Cameron). In 1994, he

graduated from medical school cum laude.

From December 1994 through December 1995, he worked as an intern at the General

Surgical Intensive Care Unit, Leiden University Medical Center (prof.dr. O.T. Terpstra).

During this time he perfonned research under the supervision afpraf.dr. H.J. van Bockel.

In February 1996, he became a resident at the Department of Surgery (chainnan prof.dr.

l. leekel), University Hospital Rotterdam 'Dijkzigt' (prof.dr. H.A. Bruining / prof.dr. H.J.

Bonjer).

From February 1999 through July 2000, he worked as a research feHow in surgery at the

Transplantation Biology Research Center, Massachusetts General Hospital I Harvard

Medical School, Boston, U.S.A. (prof.dr. D.K.C. Cooper / prof.dr. D.H. Sachs). It is at

this institution that the research outlined in this dissertation was performed. Financial

support was obtained from the 'Ter Meulen Fund' of the Royal Dutch Academy of Arts

and Sciences and from the Professor Michael van Vloten Fund.

Since August 2000, the author is completing his surgical residency at the Reinier de

GraafGasthuis in Delft (dr. P.W. de Graa!).

233

induction of immunological tolerance in the pig-to … ian patrick joseph.pdf · the inclusion...

Documents